Protein and peptide libraries

ABSTRACT

Provided herein are, inter alia, methods for linking an mRNA molecule to a polypeptide (e.g., a peptide or a protein) by linking the mRNA molecule to a linking amino acid in the polypeptide, or by linking the mRNA molecule to a linking tRNA to which the polypeptide is attached, via reactions not catalyzed by the ribosome, and methods for making polypeptide libraries. Also provided are mRNA-protein complexes and mRNA-tRNA-protein complexes, libraries containing these complexes, and methods of using these complexes.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a continuation and claims the benefit of U.S. patentapplication Ser. No. 14/236,399, filed on Jan. 31, 2014 (issued as U.S.Pat. No. 9,422,550), which is a U.S. National Phase Application under 35U.S.C. § 371 of International Patent Application No. PCT/US2012/048988,filed on Jul. 31, 2012, which claims the benefit of U.S. ProvisionalPatent Application Ser. No. 61/513,819, filed on Aug. 1, 2011, theentire contents of the foregoing are hereby incorporated by reference.

TECHNICAL FIELD

This invention relates, inter alia, to compositions and methods forgenerating and using libraries of protein and peptide molecules.

BACKGROUND

Polypeptides can adopt three-dimensional structures that are capable ofbinding to other biological molecules with very high affinity andspecificity. A library of random polypeptide sequences can be populatedby molecules with a wide variety of three-dimensional structures. Inorder to isolate a polypeptide with a conformation that interacts with aspecific target protein, individual sequences from the library can beprepared and tested or screened for their affinity to the target.However, for very large libraries (>10⁶ members), the screening ofindividual sequences for binding affinity is not feasible. To overcomethis limitation, a number of techniques have been developed to selectnovel polypeptides from extremely large, complex mixtures by virtue oftheir binding affinity to a target.

The ribosome contains two sites that are critical for the function ofaminoacylated tRNAs in protein synthesis: the peptidyl transferasecenter and the decoding site region. The mRNA display strategy developedby Szostak et al. (e.g., U.S. Pat. No. 6,258,558; and Roberts, R. W. andSzostak, J. W. (1997). Proc. Natl. Acad. Sci. USA 94:12297-12302)exploits the catalytic activity of the peptidyl transferase center tolink a 3′-puromycin derivatized mRNA to its encoded peptide by creatinga peptide bond between the two.

An mRNA can also be linked to a tRNA at the decoding center. This can beachieved through the use of naturally-occurring orartificially-introduced crosslinkers. One naturally-occurringphotochemical crosslinker is the well-known Y-base present near (withina few angstroms of) the anticodon of certain tRNAs (see, e.g., U.S. Pat.Nos. 5,843,701; 6,194,550; and 6,440,695). Others (e.g., U.S. Pat. Nos.6,962,781; 7,351,812; 7,410,761; and 7,488,600) have describedcombinations wherein a photo-activatable group on one nucleic acid canreact with a reactive group on a second nucleic acid.

SUMMARY

Provided herein are functionalized mRNA molecules, functionalized tRNAmolecules, mRNA-polypeptide complexes, mRNA-tRNA-polypeptide complexes,and methods for preparing and using mRNA display libraries.

In one aspect of the invention a linking tRNA is crosslinked to an mRNAwhile the individual elements are engaged with a ribosome. Thecrosslinking is based on introducing a specific modified nucleoside at aspecific position in the mRNA or the linking tRNA. By design, when theanticodon of the linking tRNA forms three base pairs with a codoncontaining the modified nucleoside of the mRNA, a chemical reaction caneither occur spontaneously or be induced to occur by addition of certainchemicals or, in some cases, by exposure to light. Alternatively, themodified nucleoside can be on the linking tRNA. Thus, the crosslinkingcan occur between a modified “activated” nucleoside on the mRNA and a“reactive” nucleoside within or near the anticodon of the linking tRNA,or between a modified activated nucleoside within or near the anticodonof the linking tRNA and a reactive nucleoside within or near acomplementary (or “cognate”) codon of the mRNA. In either case, thepairing of the anticodon of the linking tRNA and the complementary codonin the mRNA results in crosslinking the tRNA to the mRNA. The inventionfurther requires that the growing polypeptide chain remain covalentlylinked to the linking tRNA, and methods for accomplishing such aredescribed. If the polypeptide is covalently attached to the tRNA, andthe tRNA becomes crosslinked to the mRNA, then the polypeptide iseffectively bound covalently to the mRNA, forming anmRNA-tRNA-polypeptide complex. In some embodiments, the mRNA becomescrosslinked to an amino acid in the polypeptide, forming anmRNA-polypeptide complex. In any of the methods described herein, thepolypeptide can be viewed as being “displayed” on the mRNA. Libraries ofsuch polypeptides linked to mRNA are known as mRNA display libraries.

In one aspect, provided herein are methods for linking an mRNA moleculeto a polypeptide, these methods including: (a) providing an mRNAmolecule containing a crosslinker, wherein the crosslinker is analkylated, modified, or activated nucleoside; (b) providing atranslation system containing a linking aminoacyl-tRNA containing anamino acid residue linked to a linking tRNA by a covalent bond, whereinthe linking tRNA contains an anticodon containing a reactive nucleosidethat is reactive with the crosslinker; (c) translating the mRNA moleculein the translation system to produce a polypeptide into which the aminoacid residue, still linked to the tRNA, is incorporated; and (d) duringor after step (c), crosslinking the reactive nucleoside of the anticodonto the crosslinker of the mRNA molecule, thereby linking the mRNAmolecule to the polypeptide through the linking tRNA.

In another aspect, provided herein are methods for linking an mRNAmolecule to a polypeptide, these methods including: (a) providing anmRNA molecule containing a reactive nucleoside that is reactive with acrosslinker, wherein the crosslinker is an alkylated nucleoside; (b)providing a translation system comprising a linking aminoacyl-tRNAcontaining an amino acid residue linked to a linking tRNA by a covalentbond, wherein the linking tRNA contains an anticodon containing acrosslinker that is an alkylated nucleoside; (c) translating the mRNAmolecule in the translation system to produce a polypeptide into whichthe amino acid residue, still linked to the linking tRNA, isincorporated; and (d) during or after step (c), crosslinking thereactive nucleoside of the mRNA molecule to the crosslinker of theanticodon of the linking tRNA, thereby linking the mRNA molecule to thepolypeptide through the linking tRNA.

The alkylated nucleoside can be an alkylated guanosine. For example, thealkylated guanosine can be an N-7 alkylated guanosine. In some cases,the N-7 alkylated guanosine containsN-(2-acetamidophenyl)-2-bromoacetamide at the N-7 position of thenucleoside. The N-7 alkylated guanosine can also contain one of thefollowing at the N-7 position: (i)N-(3-acetamidophenyl)-2-bromoacetamide; (ii)N-(4-acetamidophenyl)-2-bromoacetamide; (iii)N-((2-acetamidomethyl)benzyl)-2-bromoacetamide; (iv)N-((3-acetamidomethyl)benzyl)-2-bromoacetamide; (v)N-((4-acetamidomethyl)benzyl)-2-bromoacetamide; (vi)(Z/E)-N-(4-acetamidobut-2-enyl)-2-bromoacetamide; or (vii)(Z/E)-N-(2-acetamidovinyl)-2-bromoacetamide. Reactive nucleosides thatare reactive with an alkylated guanosine include 2-thiouridine and4-thiouridine.

The alkylated nucleoside can also be an S-4-alkylated thiouridine.Guanosine, for example, is a reactive nucleoside that can react (e.g.,crosslink) with an S-4-alkylated thiouridine. In some embodiments, theS-4 alkylated thiouridine containsN-(2-acetamidophenyl)-2-bromoacetamide at the S-4 position of thenucleoside. In some embodiments, the S-4 alkylated thiouridine containsone of the following at the S-4 position: (i)N-(3-acetamidophenyl)-2-bromoacetamide; (ii)N-(4-acetamidophenyl)-2-bromoacetamide; (iii)N-((2-acetamidomethyl)benzyl)-2-bromoacetamide; (iv)N-((3-acetamidomethyl)benzyl)-2-bromoacetamide; (v)N-((4-acetamidomethyl)benzyl)-2-bromoacetamide; (vi)(Z/E)-N-(4-acetamidobut-2-enyl)-2-bromoacetamide; or (vii)(Z/E)-N-(2-acetamidovinyl)-2-bromoacetamide.

In another aspect, described herein are methods for linking an mRNAmolecule to a polypeptide, these methods including: (a) providing anmRNA molecule containing a crosslinker, wherein the crosslinker is a4-oxo-2-pentenal moiety attached to a nucleoside; (b) providing atranslation system containing a linking aminoacyl-tRNA containing anamino acid residue linked to a linking tRNA by a covalent bond, whereinthe linking tRNA contains a reactive nucleoside that is reactive withthe crosslinker; (c) translating the mRNA molecule in the translationsystem to produce a polypeptide into which the amino acid residue, stilllinked to the linking tRNA, is incorporated; and (d) during or afterstep (c), crosslinking the reactive nucleoside of the linking tRNA withthe crosslinker of the mRNA molecule, thereby linking the mRNA moleculeto the polypeptide through the linking tRNA.

In another aspect, provided herein are methods for linking an mRNAmolecule to a polypeptide, these methods including: (a) providing anmRNA molecule containing a reactive nucleoside that is reactive with acrosslinker, wherein the crosslinker is a 4-oxo-2-pentenal moietyattached to a nucleoside; (b) providing a translation system containinga linking aminoacyl-tRNA containing an amino acid residue linked to alinking tRNA by a covalent bond, wherein the linking tRNA contains acrosslinker that is a 4-oxo-2-pentenal moiety attached to a nucleoside;(c) translating the mRNA molecule in the translation system to produce apolypeptide into which the amino acid residue, still linked to thelinking tRNA, is incorporated; and (d) during or after step (c),crosslinking the reactive nucleoside of the mRNA molecule with thecrosslinker of the linking tRNA, thereby linking the mRNA molecule tothe polypeptide through the linking tRNA. In some embodiments, thecrosslinker may be within or near the anticodon on the linking tRNA andthe reactive nucleoside is in or near the complementary codon on themRNA. In some embodiments, the reactive nucleoside may be within or nearthe anticodon on the linking tRNA and the crosslinker is in or near thecomplementary codon on the mRNA.

In yet another aspect, provided herein are methods for linking an mRNAmolecule to a polypeptide, these methods including: (a) providing anmRNA molecule containing a nucleoside with a furan moiety attached; (b)providing a translation system containing a linking aminoacyl-tRNAcontaining an amino acid residue linked to a linking tRNA by a covalentbond, wherein the linking tRNA contains a reactive nucleoside that isreactive with a crosslinker, wherein the crosslinker is a4-oxo-2-pentenal moiety; (c) translating the mRNA molecule in thetranslation system to produce a polypeptide into which the amino acidresidue, still linked to the linking tRNA, is incorporated; and (d)during or after step (c), adding an oxidizing agent to oxidize the furanmoiety to generate a 4-oxo-2-pentenal moiety, and crosslinking thereactive nucleoside of the linking tRNA with the 4-oxo-2-pentenal moietyof the mRNA molecule, thereby linking the mRNA molecule to thepolypeptide through the linking tRNA. Alternatively, the furan moietymay be within or near the anticodon on the linking tRNA and the reactivenucleoside is within or near the complementary codon on the mRNA.

In another aspect, provided herein are methods for linking an mRNAmolecule to a polypeptide, these methods including: (a) providing anmRNA molecule containing a reactive nucleoside that is reactive with acrosslinker, wherein the crosslinker is a 4-oxo-2-pentenal moiety; (b)providing a translation system containing a linking aminoacyl-tRNAcontaining an amino acid residue linked to a linking tRNA by a covalentbond, wherein the linking tRNA contains a nucleoside with a furan moietyattached; (c) translating the mRNA molecule in the translation system toproduce a polypeptide into which the amino acid residue, still linked tothe linking tRNA, is incorporated; and (d) during or after step (c),adding an oxidizing agent to oxidize the furan moiety to generate a4-oxo-2-pentenal moiety, and crosslinking the reactive nucleoside of themRNA with the 4-oxo-2-pentenal moiety of the linking tRNA, therebylinking the mRNA molecule to the polypeptide through the linking tRNA.In some embodiments, the nucleoside with a furan moiety attached may bewithin or near the anticodon on the linking tRNA and the reactivenucleoside is within or near the complementary codon on the mRNA. Insome embodiments, the reactive nucleoside may be within or near theanticodon on the linking tRNA and the nucleoside with a furan moietyattached is within or near the complementary codon on the mRNA.

In the methods described herein, the 4-oxo-2-pentenal moiety on the mRNAor the linking tRNA can be generated by (i) reacting an mRNA or linkingtRNA molecule containing a 2′-amino nucleoside with anN-hydroxysuccinimide ester of a carboxyalkyl furan to generate an mRNAor tRNA product, and (ii) then treating the mRNA or tRNA product with anoxidizing agent. In some embodiments, the carboxyalkyl furan is2-(furan-3-yl) acetic acid, 3-(furan-3-yl) propanoic acid, or4-(furan-3-yl) butanoic acid. Oxidizing agents can includeN-bromosuccinimide, meta-chloro peroxybenzoic acid, methylene blue,molecular oxygen, bromine, and ultraviolet light. In some embodiments,the oxidizing agent is added during step (c).

Also provided are methods for linking an mRNA molecule to a polypeptide,these methods including: (a) providing an mRNA molecule containing acrosslinker, wherein the crosslinker is an electrophilic nucleoside thatreacts with a nucleophilic nucleoside partner viahybridization-triggered alkylation; (b) providing a translation systemcontaining a linking aminoacyl-tRNA containing an amino acid residuecovalently linked to a linking tRNA by a covalent bond, wherein thelinking tRNA contains an anticodon containing a nucleophilic nucleosidepartner; (c) translating the mRNA molecule in the translation system toproduce a polypeptide into which the amino acid residue, still linked tothe linking tRNA, is incorporated; and (d) during or after step (c),crosslinking the crosslinker of the mRNA molecule to the nucleophilicnucleoside partner of the linking tRNA, thereby linking the mRNAmolecule to the polypeptide through the linking tRNA. Alternatively, theelectrophilic nucleoside may be within or near the anticodon on thelinking tRNA and the reactive nucleoside is within or near thecomplementary codon on the mRNA.

In another aspect, provided herein are methods for linking an mRNAmolecule to a polypeptide, these methods including: (a) providing anmRNA molecule containing at a nucleophilic nucleoside partner; (b)providing a translation system containing a linking aminoacyl-tRNAcontaining an amino acid residue linked to a linking tRNA by a covalentbond, wherein the linking tRNA contains an anticodon containing acrosslinker, wherein the crosslinker is an electrophilic nucleoside thatreacts with a nucleophilic partner via hybridization-triggeredalkylation; (c) translating the mRNA molecule in the translation systemto produce a polypeptide into which the amino acid residue, still linkedto the linking tRNA, is incorporated; and (d) during or after step (c),crosslinking the crosslinker of the linking tRNA to the nucleophilicnucleoside partner of the mRNA molecule, thereby linking the mRNAmolecule to the polypeptide through the linking tRNA. In someembodiments, the crosslinker may be within or near the anticodon on thelinking tRNA and the reactive nucleoside is within or near thecomplementary codon on the mRNA. In some embodiments, the reactivenucleoside may be within or near the anticodon on the linking tRNA andthe crosslinker is within or near the complementary codon on the mRNA.

The electrophilic nucleoside and its nucleophilic nucleoside partner canbe (i) 2-amino-6-vinylpurine and cytosine; (ii) 2-amino-6-vinylpurineand 4-thiouridine; (iii) 2-alpha-halomethyl adenosine and 2-thiouridine;(iv) 8-alpha-halomethyl purine and 4-thiouridine; (v)2,8-alpha-halomethyl purine and 4-thiouridine; (vi)5-methyl-N⁴,N⁴-ethanocytosine and cytosine; (vii)2-amino-6-(1-ethylsulfinyl)vinyl purine nucleoside and cytosine; (viii)4-amino-6-oxo-2-vinylpyrimidine nucleoside and uridine; and (ix)4-amino-6-oxo-2-vinylpyrimidine-ethyl-C-nucleoside and uridine.

In another aspect, provided herein are methods for linking an mRNAmolecule to a polypeptide, these methods including: (a) providing anmRNA molecule containing a first member of a complementary crosslinkerpair; (b) providing a translation system containing a linkingaminoacyl-tRNA containing an amino acid residue linked to a linking tRNAby a covalent bond, wherein the linking tRNA contains an anticodoncontaining a second member of the complementary crosslinker pair,wherein the complementary crosslinker pair is selected from thefollowing pairs: (i) 2-thiouridine and an adenosine containing an olefinsubstitution at the 2-position of the purine ring, (ii) 4-thiouridineand 2-amino-6 vinylpurine, and (iii) 4-thiouridine and an adenosinecontaining a vinyl substituent at the 8-position of the ring; (c)translating the mRNA molecule in the translation system to produce apolypeptide into which the amino acid residue, still linked to thelinking tRNA, is incorporated; and (d) during or after step (c),crosslinking the first and second members of the complementarycrosslinker pair, thereby linking the mRNA molecule to the polypeptidethrough the linking tRNA. In this aspect of the invention, it is notcritical which member of the crosslinking pair is present on the mRNAand which is present on the linking tRNA.

Crosslinking of the first and second members of the complementarycrosslinker pair can be carried out by adding one or more of thefollowing agents: (a) iodine, (b) NBS, (c) ethanethiol and ultravioletlight, (d) bromine, and (e) meta-chloro peroxybenzoic acid.

Generally, in the methods described herein, when the crosslinker (e.g.,any of the crosslinkers described herein), reactive nucleoside,nucleoside with a furan moiety attached, nucleophilic nucleosidepartner, or first member of the complementary crosslinker pair islocated on the mRNA, it is positioned in, or within 3 nucleotides, of anin-frame stop codon on the mRNA molecule. In any of the embodimentsdescribed herein, the linking tRNA can be a suppressor tRNA. If thecrosslinker (e.g., any of the crosslinkers described herein), reactivenucleoside, nucleoside with a furan moiety attached, nucleophilicnucleoside partner, or first member of the crosslinker pair is on thelinking tRNA, then the anticodon in the linking tRNA may correspond to anonsense or stop codon, as in the case when the linking tRNA correspondsto a suppressor tRNA, or it may correspond to a sense codon, as in thecase when the linking tRNA corresponds to an elongator tRNA. Often, thesense codon corresponding to the anticodon in the linking tRNA isfollowed by one or more stop codons. In some embodiments, the mRNA orlinking tRNA molecule comprises one, two or three of the crosslinkers,reactive nucleosides, nucleosides with a furan moiety attached,nucleophilic nucleoside partners, or first members of the complementarycrosslinker pair. A purified in vitro translation system is used totranslate the mRNA molecule in some cases.

In a further aspect, provided herein are mRNA-tRNA-polypeptide complexesthat contain an mRNA covalently linked to a tRNA that is covalentlylinked to an amino acid in a polypeptide, wherein the mRNA is linked tothe tRNA via a bridging group selected from the group of: N-7alkylpurine, oxadiazabicyclo[3.3.0]octaimine, 4-aminoalkylpyrimidine,4-thioalkylpyrimidine, 2-thioalkylpyrimidine, 2-aminoalkylpyrimidine,4-alkyloxypyrimidine, an ether, a thioether, and a secondary amine. Alsoprovided herein are libraries containing a plurality of thesemRNA-tRNA-polypeptide complexes, the plurality containingmRNA-tRNA-polypeptide complexes that differ from one another, e.g.,wherein the mRNA of each mRNA-tRNA-polypeptide complex encodes adifferent polypeptide. Also provided herein are methods of screening fora polypeptide that interacts with a target, these methods including: (a)providing any of the mRNA-tRNA-polypeptide libraries described herein;(b) contacting the mRNA-tRNA-polypeptide library with the target; and(c) selecting an mRNA-tRNA-polypeptide complex containing a polypeptidethat interacts with the target.

Whereas the preceding methods for linking an mRNA to a polypeptide makeuse of a linking tRNA and crosslinking at the decoding site of theribosome, other methods do not utilize a linking tRNA. In these methods,a chemical or photochemical reaction in the vicinity of the peptidyltransfer center of the ribosome serves to covalently crosslink an mRNAto the polypeptide it encodes. In one aspect, provided herein arefunctionalized RNAs comprising an mRNA containing a coding region and,at the 3′ end of the mRNA, a 3′ substituent containing a linking moietyselected from the group of (i) a derivative of a ribo adenosinecomprising a first member of a reactive pair at its 2′ or 3′ position,(ii) a derivative of a deoxyribo adenosine containing a first member ofa reactive pair at its 3′ position, and (iii) a derivative of puromycincontaining a first member of a reactive pair, wherein the linking moietyis not capable of participating in ribosome-catalyzed peptide bondformation.

The functionalized RNAs can each include a linking moiety selected fromthe group of: a) a derivative of 2′-deoxy-2′-amino-adenosine in whichthe first member of the reactive pair is attached via an amide bond; b)a derivative of 3′-deoxy-3′-amino-adenosine in which the first member ofthe reactive pair is attached via an amide bond; c) a derivative of2′-amino-2′-3′-dideoxy-adenosine in which the first member of thereactive pair is attached via an amide bond; d) a derivative of3′-amino-2′-3′-dideoxy-adenosine in which the first member of thereactive pair is attached via an amide bond; e) a derivative ofpuromycin in which the first member of the reactive pair is attached viaan amide bond; and f) a derivative of3′-amino-3′-deoxy-N⁶,N⁶-dimethyladenosine in which the first member ofthe reactive pair is attached via an amide bond.

In some embodiments, the reactive pair is selected from the group of:(a) an azide and an alkyne; (b) an alkene and a thiol or an amine; (c) atetrazine and a trans-cyclooctane, a cyclopropene, abicyclo[2.2.1]hept-2-ene or a norbornene; (d) an α-halo-benzyl and athiol or an amine; (e) an α-halo-carbonyl and a thiol or an amine; and(f) a photocrosslinker and a moiety that reacts with thephotocrosslinker. In some embodiments, the photocrosslinker is selectedfrom the group of (a) psoralen (b) phenyl-azide derivatives; (c)phenyl-diazirine derivatives; (d) benzophenone, and (e) alkyl azides.

In some embodiments, the linking moiety of the functionalized RNA isimmediately preceded by a CC, CdC, or dCdC sequence.

In some embodiments, one of the last three codons of the coding regionof the functionalized RNAs encodes a linking amino acid that contains asecond member of the reactive pair. In some embodiments, the last codonof the coding region is a stop codon that is recognized by anaminoacylated suppressor tRNA containing a linking amino acid thatcontains a second member of the reactive pair. In some embodiments, thelinking moiety can be separated from the last codon of the coding regionby at least 30 nucleotides of RNA.

In some embodiments, the 3′ substituent of the functionalized RNAs cancontain a pause moiety between the coding region and the linking moiety.In some embodiments, the pause moiety contains a nucleic acid other thanRNA. In some embodiments, the pause moiety contains DNA, LNA, TNA, GNA,PNA, PEG, or peptide. Also provided herein are libraries containing aplurality of functionalized RNAs described herein. In some embodiments,the mRNA of each functionalized RNA of the plurality encodes a differentpolypeptide.

In another aspect, described herein are mRNA-polypeptide complexescontaining a functionalized RNA containing an mRNA, wherein thefunctionalized RNA is covalently linked via a bridging group to alinking amino acid of a polypeptide, wherein the mRNA encodes thepolypeptide and contains a codon encoding the linking amino acid, andwherein the bridging group contains triazole, thioether, secondaryamine, pyridazine, 3,4-diazanorcaradiene, benzylthioether, orbenzylamine. Also provided are libraries containing a plurality of thesemRNA-polypeptide complexes, wherein the plurality contains differentmRNA-polypeptide complexes.

In one aspect, provided herein are translation systems containing: (a) alibrary containing a plurality of functionalized RNAs; and (b) anaminoacylated tRNA containing the linking amino acid. The linking aminoacid can be selected from the group of (a) L-azidoalanine; (b)L-azidohomoalanine; (c) L-azidonorvaline; (d) 4-ethynyl-L-phenylalanine;(e) L-homopropargylglycine; (f) L-propynylglycine; (g) cysteine; and (h)lysine. In some embodiments, the aminoacylated tRNA is an aminoacylatedsuppressor tRNA. In some embodiments, the translation system is apurified translation system.

In one aspect, included herein are methods for linking an mRNA to apolypeptide. The methods including: (a) providing a functionalized RNAdescribed herein; (b) providing a translation system containing anaminoacylated tRNA containing a linking amino acid; (c) translating themRNA of the functionalized RNA to produce a polypeptide into which thelinking amino acid is incorporated; and (d) crosslinking the linkingmoiety to the linking amino acid, thereby linking the mRNA of thefunctionalized RNA to the polypeptide. The crosslinking step can becarried out by adding (a) copper, (b) UV light and ethanothiol orbeta-mercaptoenthanol, (c) aqueous iodine or bromine, or (d) UV light.

In one aspect, methods of generating a library of mRNA-polypeptidecomplexes are provided. These methods include: (a) providing a libraryof functionalized RNAs (a library of functionalized RNAs as describedherein); (b) providing a translation system containing an aminoacylatedtRNA containing the linking amino acid; (c) translating the mRNAs of theplurality of functionalized RNAs to produce a plurality of diversepolypeptides into each of which the linking amino acid is incorporated;and (d) crosslinking the linking moiety of each functionalized RNA tothe linking amino acid incorporated into the polypeptide translated fromthat functionalized RNA, thereby linking each mRNA of the plurality offunctionalized RNAs to the polypeptide it encodes and generating alibrary of mRNA-polypeptide complexes.

In another aspect, provided herein are methods of screening for apolypeptide that interacts with a target, these methods including: (a)providing a library of the mRNA-polypeptide complexes described herein;(b) contacting the library with the target; and (c) selecting anmRNA-polypeptide complex containing a polypeptide that interacts withthe target.

The details of a number of embodiments are set forth in the accompanyingdrawings and the description below. Other features, objects, andadvantages of the disclosed compositions and methods will be apparentfrom the description and drawings, and from the claims.

DESCRIPTION OF DRAWINGS

FIG. 1 shows an exemplary strategy for linking an mRNA molecule to apolypeptide encoded by the mRNA. In this diagram, the reactive base pairis on the mRNA, but it may be present on the linking tRNA also, with thesame final result. Furthermore, in this diagram the covalent bondbetween the linking tRNA and the amino acid is an amide. It isappreciated by those skilled in the art that other types of covalentbonds, including an ester bond, can be used so long as reasonable careis taken to preserve the bridging group (e.g., avoiding prolongedexposure to elevated temperature, exposure to acidic or basic conditionsincompatible with ester bonds).

The diagram shows the ribosome during translation with the peptidyl-tRNAand aa-tRNA shown prior to the peptide transferase reaction (the aa-tRNAwill be the C-terminal amino acid of the peptide) (left). The inset inthe diagram shows the triplet codon-anticodon pairing, with the central,reactive base on the mRNA. The diagram shows that the growing peptidechain is transferred to the terminal aa-tRNA, and the new peptidyl-tRNAand the mRNA translocates within the ribosome (immediately to the rightof the inset). The right part of the diagram shows that after removal ofthe ribosome, the peptide and mRNA remain attached due to the bases inthe codon (1) and anticodon, and the unstable link between the peptideand the tRNA (2).

FIG. 2 depicts (A) the structures of various alkylated nucleosides and(B) an example of the crosslinking reaction between an alkylatednucleoside and a complementary reactive nucleoside. In (B),4-thiouridine is first reacted withN,N′-bis-(bromoacetyl)-o-phenylenediamine (left). Upon base-pairing witha complementary guanosine, the remaining free alkylating group reactswith the N7 position of the guanosine, with the reaction promoted byhydrogen bonding between the guanosine C6 carbonyl group and the amidehydrogens of the reagent (center). The crosslinked product is shown onthe right.

FIG. 3 shows the incorporation of a furan residue into anoligonucleotide and subsequent oxidation with N-bromosuccinimde (NBS),which leads to crosslinking to an adenosine or cytidine residue on theopposite strand.

FIGS. 4A and 4B show examples of pairs of an electrophilic nucleosideand its nucleophilic nucleoside partner: (i) 2-amino-6-vinylpurine andcytosine, (ii) 2-amino-6-vinylpurine and 4-thiouridine, (iii)2-thiouridine and 2-alpha-halomethyl adenosine, (iv) 4-thiouridine and8-alpha-halomethyl purine, (v) 4-thiouridine and 2,8-alpha-halomethylpurine, (vi) 5-methyl-N⁴,N⁴-ethanocytosine and cytosine, (vii)2-amino-6-(1-ethylsulfinyl)vinyl purine nucleoside and cytosine, (viii)4-amino-6-oxo-2-vinylpyrimidine nucleoside and guanine, and (ix)4-amino-6-oxo-2-vinylpyrimidine-ethyl-C-nucleoside and uridine. R can beribose, deoxyribose, or any suitable sugar (such as threose or glycerol)or a functional element (such as an amino acid) that is capable offorming a polymer.

FIG. 5 depicts examples of complementary crosslinker pairs. Top:2-thiouridine and an adenosine containing an olefin substitution at the2-position of the purine ring. Middle: 4-thiouridine and2-amino-6-vinylpurine. Bottom: 4-thiouridine and an adenosine containinga vinyl substituent at the 8-position of the purine ring.

FIG. 6 illustrates the steps in an exemplary method for preparing a2-amino-6-vinylpurine phosphoramidite.

FIG. 7 shows the steps in an exemplary method for synthesizingoligonucleotides containing 2-amino-6 vinylpurine. The oligonucleotidescan be ligated to an mRNA or portions of a linking tRNA molecule toproduce reactive molecules suitable for practicing the presentinvention. (TOM: (triisopropylsilyl)oxy]methyl; MMPP: magnesiummonoperphthalate; and TBAF: tetra-n-butylammonium fluoride.) FIGS. 8A-Eshow exemplary linking moieties. FIG. 8A shows linking moietiescontaining an alkyne group; FIG. 8B shows linking moieties containing analkene group;

FIG. 8C shows linking moieties containing an azide group; FIG. 8D showslinking moieties containing a tetrazine group; and FIG. 8E shows linkingmoieties containing a photocrosslinker.

FIGS. 9A-C show exemplary bridging groups created between an mRNA and apolypeptide using different reactive pairs. FIG. 9A shows reactive pairsof an azide and an alkyne; FIG. 9B shows reactive pairs of an alkene anda thiol (first and middle) or an amine (bottom); FIG. 9C shows reactivepairs of a tetrazine and a cyclopropene (top), a trans-cyclooctene(middle), or a norbornene (bottom).

FIGS. 10A-C show exemplary 3′-terminal nucleotides containing an aminoresidue that can be modified with the different linking moieties shownin FIGS. 8A-E, or used to produce the functionalized RNAs describedherein. FIG. 10A shows 2′-deoxy-2′-amino-adenosine (1),3′-deoxy-3′-amino-adenosine (2), 2′-amino-2′-3′-dideoxy-adenosine (3),and 3′-amino-2′-3′-dideoxy-adenosine (4); FIG. 10B shows puromycin; andFIG. 10C shows 3′-amino-3′-deoxy-N⁶,N⁶-dimethyladenosine.

DETAILED DESCRIPTION

Described herein are tRNA molecules, mRNA molecules encoding apolypeptide, as well as novel methods for linking mRNA molecules to thepolypeptides (e.g., a peptide or a protein) encoded by the mRNAmolecules. The methods can be used to create vast libraries ofpolypeptides from which those with desired target binding or othertarget-specific activities can be selected along with their encodingmRNA. The invention can also be applied to in vitro evolution ofpolypeptides in order to optimize their binding affinities or otherproperties.

Methods of Linking an mRNA Molecule to a Polypeptide

Two general approaches are described to link a polypeptide to itsencoding mRNA. In one method, the mRNA is indirectly linked to thepolypeptide through a linking tRNA. In a second method, the mRNA islinked to the polypeptide through a chemical or photochemical reactionbetween the terminus of the mRNA molecule and a portion of thepolypeptide chain.

Linking a Polypeptide to an Encoding mRNA Through a tRNA

In one approach for linking an mRNA molecule to a polypeptide, the mRNAmolecule and the polypeptide become linked through a tRNA, forming anmRNA-tRNA-polypeptide complex. A tRNA that links an mRNA to apolypeptide is referred to herein as a “linking tRNA.”

As used herein, by “a” is meant at least one. As used herein, anucleoside includes a nucleobase (or “base”) bound to a ribose ordeoxyribose sugar. A nucleoside can be a naturally-occurring nucleoside(i.e., cytosine, adenosine, guanosine, thymidine, or uridine) or amodified nucleoside that differs from a naturally-occurring nucleosidein the structure of its nucleobase and/or sugar. The nucleobase can bebound to other sugars, such as threose, or other polymer-formingmolecules, such as glycerol or an amino acid.

Generally, the methods involve using an mRNA or a linking tRNA moleculethat contains at least one (e.g., two, three, or four) crosslinker orreactive nucleoside that can crosslink with a nucleoside (e.g., anatural or modified nucleoside that is reactive with the crosslinker orreactive nucleoside) in a linking tRNA or an mRNA, respectively. Theterm “crosslinker” refers to any moiety that can be used to covalentlylink two molecules together via a chemical reaction between (a) themoiety on one molecule and (b) a second molecule. In non-limitingexamples, a crosslinker can be a modified nucleoside or a moiety on anatural or modified nucleoside. Crosslinking between two molecules canoccur spontaneously, or by adding an agent or performing a treatmentthat induces crosslinking. Additional examples of crosslinkers aredescribed herein. A reactive nucleoside can include any of the naturalor modified nucleosides described herein.

An mRNA (e.g, one containing at least one crosslinker or reactivenucleoside) described herein is translated to produce a polypeptideusing an in vitro translation system, as described in more detail below.A linking aminoacyl-tRNA includes a linking tRNA and an aminoacylresidue attached to the linking tRNA by a stable, non-hydrolyzablecovalent bond. An example of such a bond is an amide bond. Those skilledin the art will appreciate that a less stable ester bond may also beused as long as measures are taken to protect it from hydrolysis (e.g.,avoiding prolonged exposure to elevated temperatures or acidic or basicconditions that are incompatible with an ester bridging group). If themRNA does not contain a crosslinker or reactive nucleoside), then thecrosslinker or reactive nucleoside is present on the linking tRNA. Afterthe linking aminoacyl-tRNA accepts the nascent polypeptide by the actionof the ribosome's peptidyl transferase, the nascent polypeptide willremain attached to the linking tRNA. Upon crosslinking of thecrosslinker or reactive nucleoside in the mRNA to a nucleoside in thelinking tRNA, or upon crosslinking of the crosslinker or reactivenucleoside in the linking tRNA to a nucleoside in the mRNA, the linkingtRNA and the mRNA become covalently linked, and a covalent linkage ofthe polypeptide to the mRNA through the linking tRNA is created. FIG. 1illustrates an exemplary strategy for linking an mRNA molecule to apolypeptide encoded by the mRNA. In FIG. 1, the crosslinker (e.g., thereactive base) is shown as positioned in the mRNA, but it may be presenton the linking tRNA instead.

Generally, when a crosslinker or reactive nucleoside is in the mRNA, itis positioned at (or within one to three bases from) the end of thepolypeptide coding region of the mRNA molecule. The crosslinker orreactive nucleoside can be positioned in an in-frame codon encoding anamino acid or in an in-frame stop codon, or can be in a nucleotide thatis within one to three bases 3′ to a stop codon at the end of the codingregion. In most cases, crosslinking between the crosslinker or reactivenucleoside in the mRNA and a nucleoside in the linking tRNA requiresspecific pairing between the codon containing the crosslinker orreactive nucleoside and its corresponding anticodon in the linking tRNAduring translation of the mRNA. In other cases, as described herein, thecrosslinker or reactive nucleoside does not have to be in a codonrecognized by the anticodon of the linking tRNA. For example, thecrosslinker or reactive nucleoside can be positioned near (e.g., withinone to three bases from) a codon in the mRNA that corresponds to theanticodon of the linking tRNA. In some embodiments, the mRNA molecule orthe linking tRNA can include more than one (e.g. two, three, or four) ofthe same crosslinker, or multiple different crosslinkers. Optimizedconfigurations of the number and/or positions of crosslinkers can bedetermined by creating mRNAs or linking tRNAs with differentconfigurations and assaying the yield of crosslinkedmRNA-tRNA-polypeptide complexes, e.g., by electrophoresis.

A linking tRNA can contain one or more (e.g., two, three, or four)natural or modified nucleosides (e.g., activated nucleosides) orcrosslinkers that can react with a nucleoside (e.g., a reactivenucleoside) in an mRNA molecule, such that the linking tRNA can becovalently linked to the mRNA molecule. A reactive nucleoside can be anucleoside that is reactive with an activated or modified nucleoside, orcrosslinker. Generally, but not always, the nucleoside or crosslinkerthat can react with a crosslinker or a nucleoside on the mRNA ispositioned in the anticodon of the linking tRNA. The linking tRNA can bea native, modified, or synthetic tRNA. The anticodon of the linking tRNAcan be one that recognizes a sense or stop codon. In some embodiments,the linking tRNA recognizes a sense codon that immediately precedes oneor more stop codons. In some embodiments, the linking tRNA recognizes astop codon. The term “suppressor tRNA” refers to a tRNA that recognizesa stop codon. In some embodiments, the linking tRNA contains at leastone (e.g., two, three, or four) activated nucleoside (e.g., acrosslinker or modified nucleoside) and the mRNA contains at least one(e.g., two, three, or four) natural or modified reactive nucleosidesthat can crosslink to at least one (e.g., one, two, three, or four)activated nucleoside in the linking tRNA. In other embodiments, theanticodon of the linking tRNA is a four-base sequence that recognizes afour-base codon in an mRNA. When the crosslinker is located in ananticodon of a linking tRNA that encodes an amino acid, the mRNA can bedesigned such that the corresponding codon only occurs once in the mRNA(i.e., at the site at which crosslinking is desired).

The methods described herein allow the production ofmRNA-tRNA-polypeptide complexes. Non-limiting examples ofmRNA-tRNA-polypeptide complexes that are provided herein contain an mRNAcovalently linked to a tRNA that is covalently linked to an amino acidin a polypeptide, where the mRNA is linked to the tRNA via a bridginggroup selected from the group of: N-7 alkylpurine,oxadiazabicyclo[3.3.0]octaimine, 4-aminoalkylpyrimidine,4-thioalkylpyrimidine, 2-thioalkylpyrimidine, 2-aminoalkylpyrimidine,4-alkyloxypyrimidine, an ether, a thioether, and a secondary amine. Alsoprovided herein are libraries containing a plurality of thesemRNA-tRNA-polypeptide complexes, where the mRNA of eachmRNA-tRNA-polypeptide complex encodes a different polypeptide. In someembodiments, libraries containing a plurality of thesemRNA-tRNA-polypeptide complexes contain different mRNA-tRNA-polypeptidecomplexes. These libraries can be used to screen for a polypeptide thatinteracts with a target. Non-limiting examples of screening methods aredescribed herein. An example of such screening methods include: (a)providing any of the libraries containing a plurality ofmRNA-tRNA-polypeptide complexes described herein; (b) contacting thelibrary with the target; and (c) selecting an mRNA-tRNA-polypeptidecomplex containing a polypeptide that interacts with the target.Additional methods of using mRNA-tRNA-polypeptide complexes aredescribed herein and may be used in any combination without limitation.

Crosslinking between an Alkylated Nucleoside and its Reactive Nucleoside

One method for covalently linking an mRNA molecule to a linking tRNAinvolves crosslinking at least one (e.g., two, three, or four) alkylatednucleoside in the mRNA to at least one (e.g., two, three, or four)reactive nucleoside (e.g., one that reacts with the alkylatednucleoside) positioned in the anticodon of the linking tRNA. An exampleof the crosslinking reaction between an alkylated nucleoside and itsreactive nucleoside is illustrated in FIG. 2. Crosslinking of analkylated nucleoside to its reactive nucleoside occurs spontaneously,e.g., without requiring the addition of an agent or treatment to inducecrosslinking. Preferably, the alkylated nucleoside and its reactivenucleoside are complementary nucleosides so as to take advantage of thegeometry of their normal hydrogen bond pairing to increase the effectivemolarity and drive the reaction to completion. Inter-strand crosslinkingof a 4-thiouridine residue with a complementary guanosine residue in DNAhas been described (see, Coleman, R. S. and Kesicki, E. A., J. Org.Chem. 60:6252-6253, 1995; and Coleman, R. S. and Pires, R. M., Nucl.Acids. Res. 25:4771-4777, 1997). Alternatively, the alkylated nucleosidecan be in the anticodon of the linking tRNA and the reactive nucleosidecan be in the mRNA (e.g., in the corresponding codon).

Those of ordinary skill in the art would understand that there are manypossible combinations of an alkylated nucleoside and its reactivenucleoside. For example, an alkylated 4-thiouridine can be crosslinkedto its reactive nucleoside, such as guanosine. Alternatively, analkylated guanosine can be crosslinked to its reactive nucleoside, suchas 4-thiouridine. The alkylated guanosine can be an N-7 alkylatedguanosine. The N-7 alkylated guanosine can contain, for example, one ofthe following at the N-7 position of the guanosine nucleoside:N-(2-acetamidophenyl)-2-bromoacetamide,N-(3-acetamidophenyl)-2-bromoacetamide,N-(4-acetamidophenyl)-2-bromoacetamide,N-((2-acetamidomethyl)benzyl)-2-bromoacetamide,N-((3-acetamidomethyl)benzyl)-2-bromoacetamide,N-((4-acetamidomethyl)benzyl)-2-bromoacetamide,(Z/E)-N-(4-acetamidobut-2-enyl)-2-bromoacetamide, or(Z/E)-N-(2-acetamidovinyl)-2-bromoacetamide.

In some embodiments, an S-4-alkylated thiouridine and its reactivenucleoside, e.g., guanosine, can be used to link the mRNA to the linkingtRNA. The S-4 alkylated uridine can include one of the following at theS-4 position of the nucleoside: N-(2-acetamidophenyl)-2-bromoacetamide,N-(3-acetamidophenyl)-2-bromoacetamide,N-(4-acetamidophenyl)-2-bromoacetamide,N-((2-acetamidomethyl)benzyl)-2-bromoacetamide,N-((3-acetamidomethyl)benzyl)-2-bromoacetamide,N-((4-acetamidomethyl)benzyl)-2-bromoacetamide,(Z/E)-N-(4-acetamidobut-2-enyl)-2-bromoacetamide, or(Z/E)-N-(2-acetamidovinyl)-2-bromoacetamide.

An mRNA containing an alkylated nucleoside can be generated usingmethods known in the art and described herein (e.g., in Example 1below). Typically, the mRNA molecule contains one to three alkylatedresidues (e.g., at the 3′ end of the mRNA or at the 3′ end of thepolypeptide coding region) that can crosslink with one to three reactivenucleosides in the anticodon of the linking tRNA.

Whichever combination of alkylated nucleoside and reactive nucleoside ischosen, it is understood that the alkylated nucleoside(s) can be presenton the linking tRNA and a suitable reactive nucleoside(s) can be presenton the mRNA.

Crosslinking Using a 4-oxo-2-Pentenal Moiety

Another method for linking a coding molecule to a linking tRNA involvescrosslinking at least one (e.g., two, three, or four) 4-oxo-2-pentenalmoiety in the mRNA to at least one (e.g., two, three, or four)nucleoside, typically an adenosine or a cytidine, in the linking tRNA.This method does not require specific pairing between a nucleosidecontaining a 4-oxo-2-pentenal moiety and a complementary nucleoside inthe anticodon of the linking tRNA. For example, the 4-oxo-2-pentenalmoiety can be attached to a nucleoside positioned near (e.g., within oneor two bases of) a codon that is recognized by the anticodon (whichcontains an adenosine or a cytidine) of the linking tRNA. In anotherpossible configuration, the nucleoside containing a 4-oxo-pentenalmoiety is in a codon recognized by the anticodon of the linking tRNA,and the adenosine or cytidine residue that can react with the4-oxo-pentenal moiety is positioned adjacent to the anticodon (e.g., ina nucleoside that is one or two residues away from the anticodon).

An mRNA containing a 4-oxo-2-pentenal moiety can be created by firstreacting an mRNA molecule containing, for example, a 2′-amino nucleosidewith an N-hydroxysuccinimide ester of an alkyl furan to incorporate afuran moiety into the mRNA, and then treating the mRNA with an oxidizingagent to generate a 4-oxo-2-pentenal moiety on the mRNA. Alkyl furanswith different linker sizes can be used. For example, the alkyl furancan be 2-(furan-3-yl) acetic acid, 3-(furan-3-yl) propanoic acid, or4-(furan-3-yl) butanoic acid. Oxidizing agents that can be used include,but are not limited to, N-bromosuccinimide (NBS), meta-chloroperoxybenzoic acid, methylene blue, molecular oxygen, bromine, andultraviolet light.

Once a 4-oxo-2-pentenal moiety is created on an mRNA or tRNA, the moietycan spontaneously react (e.g., without adding an agent or treatment toinduce the reaction) with an adenosine or a cytidine in the linking tRNAor mRNA during or after translation of the mRNA. Thus, an mRNA or tRNAmolecule with at least one 4-oxo-2-pentenal moiety can be created beforethe mRNA is translated using a translation system. Alternatively, atranslation system including an oxidizing agent can be used to translatean mRNA molecule that comprises a furan moiety to create a4-oxo-pentenal moiety on the mRNA during translation. In yet anotheroption, the oxidizing agent can be added during or after translation ofan mRNA containing a furan moiety.

The 4-oxo-2-pentenal moiety can also be present on the linking tRNA suchthat it reacts with an adenosine or a cytidine in the mRNA. This methoddoes not require specific pairing between a nucleoside containing a4-oxo-2-pentenal moiety and a complementary nucleoside in thecorresponding codon of the mRNA. The 4-oxo-2-pentenal moiety can beattached to a nucleoside positioned near (e.g., within one or two basesof) the anticodon of the linking tRNA that corresponds to a codon of themRNA. In another possible configuration, the nucleoside containing a4-oxo-pentenal moiety is in the anticodon of the linking tRNA, and theadenosine or cytidine residue that can react with the 4-oxo-pentenalmoiety is positioned adjacent to corresponding codon of the mRNA (e.g.,in a nucleoside that is one or two residues away from the codon).

A linking tRNA containing at least one 4-oxo-2-pentenal moiety can becreated by first reacting a linking tRNA molecule or a portion of alinking tRNA molecule comprising, for example, a 2′-amino nucleosidewith an N-hydroxysuccinimide ester of an alkyl furan to incorporate afuran moiety into the linking tRNA or portion thereof, and then treatingthe linking tRNA or portion thereof with an oxidizing agent to generatea 4-oxo-2-pentenal moiety on the linking tRNA or portion thereof. Alkylfurans with different linker sizes can be used. For example, the alkylfuran can be 2-(furan-3-yl) acetic acid, 3-(furan-3-yl) propanoic acid,or 4-(furan-3-yl) butanoic acid. Oxidizing agents include, but are notlimited to, N-bromosuccinimide (NBS), meta-chloro peroxybenzoic acid,methylene blue, molecular oxygen, bromine, and ultraviolet light.

As described above for 4-oxo-2-pentenal-modified mRNA, a linking tRNAmolecule with at least one 4-oxo-2-pentenal moiety can be created beforethe linking tRNA is used in a translation system. Alternatively, atranslation system including an oxidizing agent can be used to convert alinking tRNA molecule containing a furan moiety to a 4-oxo-pentenalmoiety during translation, or the oxidizing agent can be added aftertranslation.

An illustration of exemplary reactions involved in using a4-oxo-2-pentenal moiety to link an mRNA molecule to a linking tRNA isshown in FIG. 3.

Crosslinking between an Electrophilic Nucleoside and its NucleophilicNucleoside Partner Via Hybridization Triggered Alkylation

Yet another strategy for linking an mRNA molecule to a linking tRNAinvolves crosslinking between an electrophilic nucleoside and itsnucleophilic nucleoside partner via hybridization-triggered alkylation.As used herein, “hybridization-triggered alkylation” refers to thecovalent linking of two nucleosides, one containing an electrophilicnucleobase and the other containing a nucleophilic nucleobase, thatoccurs as a result of the two nucleosides interacting via non-covalenthydrogen bonding. See, Webb, T. R. and Matteucci, M. D., Nucl. Acids.Res. 14:7661-7674, 1986; Webb, T. R. and Matteucci, M. D., J. Am. Chem.Soc. 108:2764-2765, 1986. Thus, an electrophilic nucleoside and itsnucleophilic nucleoside partner will frequently be complementarynucleobases. Those of ordinary skill in the art will appreciate that theelectrophilic nucleoside can be in the mRNA and its nucleophilicnucleoside partner can be in the anticodon of the linking tRNA, or viceversa. FIG. 4 shows examples of electrophilic nucleosides and theirnucleophilic nucleoside partners.

Pairs of electrophilic and nucleophilic nucleoside partners include, butare not limited to, 2-amino-6-vinylpurine and cytosine,2-amino-6-vinylpurine and 4-thiouridine, 2-alpha-halomethyl adenosineand 2-thiouridine, 8-alpha-halomethyl purine and 4-thiouridine,2,8-alpha-halomethyl purine and 4-thiouridine,5-methyl-N⁴,N⁴-ethanocytosine and cytosine;2-amino-6-(1-ethylsulfinyl)vinyl purine nucleoside and cytosine,4-amino-6-oxo-2-vinylpyrimidine nucleoside and guanine, and4-amino-6-oxo-2-vinylpyrimidine-ethyl-C-nucleoside and uridine.

An mRNA molecule containing at least one (e.g., two, three, or four)electrophilic nucleoside can be generated using methods known in the artor described herein. For example, Example 5 describes the synthesis of2-amino-6-vinylpurine phosphoramidites which can be incorporated intooligonucleotides as described in Example 6. The oligonucleotides canthen be ligated to mRNA molecules to produce templates for translation,or the oligonucleotides can be ligated to fragments of tRNA molecules toform functional linking tRNA molecules, using methods known to oneskilled in the art or described herein (see, e.g., Example 7).

Crosslinking between Two Members of a Complementary Crosslinker Pair

Another strategy for linking an mRNA molecule to a linking tRNA involvescrosslinking between members of a complementary crosslinker pair (eachmember being a natural or modified nucleoside) that can react with eachother, spontaneously or when an agent or treatment is added. Thus, anmRNA or linking tRNA molecule containing one or more moieties comprisingat least one (e.g., two, three, or four) member of the complementarycrosslinking pair can be linked to a linking tRNA or mRNA, respectively,containing at least one (e.g., two, three, or four) of the other memberof the complementary crosslinking pair. This strategy generally requiresthat the anticodon of the linking tRNA contains at least one (e.g., two,three, or four) member of the complementary crosslinking pair andcorresponds to the codon of the mRNA containing the at least one (e.g.,two, three, or four) of the other member of the complementarycrosslinking pair. Exemplary complementary crosslinker pairs and theircrosslinking reactions are illustrated in FIG. 5.

One approach is to use, as one member of the pair, a modified purinenucleoside containing an olefin at the 2-position of the purine ring.The double bond of the olefin can be selectively attacked by iodine toform an intermediate that could alkylate a thiol group in its proximity.This reaction has been demonstrated to occur in aqueous solution in anintramolecular format. See, Mizutani, M. and Sanemitsu, Y., J. Org.Chem. 50:764-768, 1985. The olefin can be synthesized viapalladium-catalyzed coupling of 2-iodoadenosine (Nair, V. et al., J.Chem. Soc., Chem. Commun. 878-879, 1989; and Nair, V. and Buenger, G.S., J. Am. Chem. Soc. 111:8502-8504, 1989), and the resulting nucleosidecan then be phosphorylated and ligated to an oligonucleotide toconstruct an mRNA with the desired modification. Another option is tosynthesize 2-iodoadenosine-containing oligonucleotides via solid phasesynthesis on CPG resin, and then perform the vinyl coupling while on theCPG resin, before deprotection. This synthesis strategy has beenpreviously used to make 2-alkynyladenosine derivatives (Piton, N. etal., Nucl. Acids Res. 35:3128-3143, 2007). A complementary crosslinkerpair can include, for example, 2-thiouridine and an adenosine containingan olefin substitution at the 2-position of the purine ring. An mRNAcontaining an olefin is translated using a translation system containinga linking tRNA with an anticodon including a 2-thiouridine. Crosslinkingbetween the 2-thiouridine and the olefin can be achieved by the additionof iodine, NBS, or ethanethiol and ultraviolet light, which forms analkyl halide that reacts with the 2-thiouridine.

Other examples of complementary crosslinker pairs include (a)4-thiouridine and 2-amino-6 vinylpurine, and (b) 4-thiouridine and anadenosine containing a vinyl substituent at the 8-position of the ring.In these cases, crosslinking between the 4-thiouridine and the vinylgroup can be achieved by the addition of iodine, NBS, or ethanethiol andultraviolet light, which forms an alkyl halide that reacts with the4-thiouridine.

Directly Linking a Polypeptide to an mRNA

In a second approach for linking an mRNA molecule to a polypeptide, theterminus of a functionalized mRNA molecule is directly linked to thepolypeptide forming an mRNA-polypeptide complex.

In this approach, the functionalized RNA described herein contains anmRNA, and at the 3′ end of the mRNA, a 3′ substituent that includes alinking moiety. The linking moiety is capable of entering theaminoacyl-tRNA binding site (A site) of the ribosome during translationof the mRNA, but lacks a peptide bond acceptor moiety that canparticipate in ribosome catalyzed peptide bond formation. Instead, thelinking moiety includes one member of a reactive pair that can reactwith and become crosslinked to another member of the reactive pairlocated on the side chain of a linking amino acid of the nascentpolypeptide. As used herein, a reactive pair is a pair of moieties thatcan crosslink with each other to form a covalent bond via a chemicalreaction not catalyzed by the ribosome. The crosslinking reaction canoccur spontaneously, or by adding an agent or performing a treatmentthat induces crosslinking. A functionalized RNA containing an mRNA and alinking moiety described herein is translated to produce a polypeptideusing an in vitro translation system (as described in more detail below)containing an aminoacyl-tRNA containing the linking amino acid. When thelinking moiety occupies the A site of the ribosome, its proximity to theP site of the ribosome allows the member of the reactive pair that ispart of the linking moiety to react with the other member of thereactive pair that is on the side chain of the linking amino acid thathas been incorporated into the growing polypeptide chain and is near theP site. Upon crosslinking between the two members of the reactive pair,the mRNA of the functionalized RNA becomes linked to the polypeptide viathe newly created covalent bond between the linking moiety and thelinking amino acid.

Functionalized RNAs and Methods of Making and Using them

A functionalized RNA includes an mRNA and one or more non-RNAcomponents. As used herein, an mRNA refers to an RNA comprising apolypeptide coding region and RNA sequences required for translation ofthe polypeptide. An mRNA can also contain other RNA sequences, such asspacer sequences and primer binding sequences used for PCR amplificationof the mRNA. Generally, the functionalized RNA described hereincontains, at the 3′ end of the mRNA, a 3′ substituent containing alinking moiety. In some embodiments, the 3′ substituent is made upentirely of the linking moiety, so that the linking moiety is directlyattached to the 3′ end of the mRNA. In other embodiments, the 3′substituent includes other moieties (e.g., non-RNA molecules) positionedbetween the linking moiety and the 3′ end of the mRNA.

One example of a linking moiety is designed based on the high affinityof the ribosome for the CCA trinucleotide (the structure of which mimicsthe release factor on stalled ribosomes). The CCA trinucleotide canenter the A site of the ribosome, but cannot participate in ribosomecatalyzed peptide bond formation. Thus, the 3′ substituent can include aribo-adenosine derivative having a member of a reactive pair at its 2′or 3′ position or a deoxyribo-adenosine derivative having a member of areactive pair at its 3′ position. Preferably, the derivative of aribo-adenosine or a deoxyribo-adenosine containing a member of areactive pair is preceded immediately by a CC or dCdC sequence. Theadenosine derivative, as exemplified in FIG. 10A, can be a derivative of2′-deoxy-2′-amino-adenosine (1), 3′-deoxy-3′-amino-adenosine (2),2′-amino-2′-3′-dideoxy-adenosine (3), or3′-amino-2′-3′-dideoxy-adenosine (4), each including a member of areactive pair that is preferably attached at the amino group via astable covalent bond (e.g., an amide bond).

A linking moiety can also be a derivative of puromycin (see FIG. 10B)that lacks puromycin's peptide bond acceptor moiety (an NH₂ group) andcontains a member of a reactive pair. Such a puromycin derivative canstill bind to the A site of a ribosome, but cannot participate inribosome-catalyzed bond formation. For example, the peptide bondacceptor moiety of puromycin can be replaced by a member of a reactivepair. An example of a linking moiety that is a derivative of puromycinis N-3′-(2-azido-3-(4-methoxyphenyl)propanamide)-3′-deoxy-N⁶,N⁶-dimethyladenosine. Puromycin derivativessuch as 3′-amino-3′-deoxy-N⁶,N⁶-dimethyladenosine (see FIG. 10C), withits peptide bond acceptor moiety removed and containing a member of areactive pair, can also be used as a linking moiety.

The member of a reactive pair that can be part of the linking moietycould be, for example, an alkyne, an azide, an alkene, a tetrazine, analpha-halo-benzyl, an alpha-halo-carbonyl, or a photocrosslinker. Analkyne (see FIG. 8A) or an alkene (see FIG. 8B) of any linker length canbe used. Azide-reactive moieties can include any azide, for example, analkyl azide of any linker length (see FIG. 8C). A tetrazine-reactivemoiety can include, for example, a 1,2,4,5-tetrazine with an aryl ringor alkyl chain (see FIG. 8D). Alpha-halo-benzyl moieties can includebenzyl bromide and benzyl chloride. Examples of alpha-halo-carbonylmoieties include bromoacetate and chloroacetate. Photocrosslinkers(e.g., moieties that can crosslink with other moieties upon activationby light) known in the art can be part of the linking moieties. Examplesof photocrosslinkers include psoralen, phenyl azide derivatives,phenyl-diazirine derivatives, benzophenone, and alkyl azides (see FIG.8E).

Exemplary reactive pairs include (a) an azide and an alkyne; (b) analkene and a thiol or an amine; (c) a tetrazine and a trans-cyclooctane,a cyclopropene, a bicyclo[2.2.1]hept-2-ene or a norbornene; (d) anα-halo-benzyl and a thiol or an amine; (e) an α-halo-carbonyl and athiol or an amine; and (f) a photocrosslinker and a moiety that reactswith the photocrosslinker. Photocrosslinkers can react non-specificallywith many moieties (e.g., alkyl rings and chains, aromatic compounds,heterocycles and alkyl chains containing heteroatoms) on the side chainof an amino acid. In general, it is not critical which member of thereactive pair is part of the linking moiety and which one is part of thelinking amino acid.

A functionalized RNA described herein can be generated by reacting anmRNA molecule containing, at its 3′ end, a 3′ substituent containing (a)a 2′ or 3′ amino ribo-adenosine, (b) a 3′ amino deoxy-ribo-adenosine, or(c) puromycin or derivative thereof, with an acylating agent, such as anN-hydroxysuccinimide (NHS) ester of the reactive moiety. The primaryamine group of the adenosine sugar residue, or puromycin or derivativethereof, reacts with the NHS ester to attach, via an amide bond, thereactive moiety to the 3′ substituent, thereby forming a linking moietyas part of the 3′ substituent. Table 1 lists exemplary acylating agentsfor adding a reactive moiety to the 3′ substituent on a functionalizedRNA. An mRNA molecule having at its 3′ end a 3′ substituent containing a2′ or 3′ amino adenosine residue, or puromycin or derivative thereof,can be made using methods known in the art, e.g., Eisenhuth, R. andRichert C., J. Org. Chem. (2009) 74, 26-37; Moroder et al., Angew. Chem.Int. Ed. (2009) 48, 4056-4060).

The mRNA of the functionalized RNA includes a codon encoding the linkingamino acid. This codon is located within the 3′ terminal portion of thepolypeptide coding region of the mRNA. In some embodiments, the codonencoding the linking amino acid is one of the last three codons of thecoding region. In some cases, the codon encoding the linking amino acidis the last or the second to the last codon of the coding region. Thecodon encoding the linking amino acid can be a sense codon or anon-sense codon (e.g., stop codon). When the codon encoding the linkingamino acid is a non-sense codon, the aminoacylated tRNA containing thelinking amino acid is an aminoacylated suppressor tRNA (i.e., one thatrecognizes a non-sense codon).

In some embodiments, the 3′ substituent contains only the linkingmoiety, so the linking moiety is located right at the 3′ end of themRNA. The mRNA can also include a spacer RNA sequence between the lastcodon of the coding region and the 3′ substituent. The spacer sequencecan include 1-30 nucleotides, e.g., 1-5, 5-15, 15-20, or 20-25nucleotides. It may be useful to include a spacer sequence with at least10 nucleotides, but no more than 300 nucleotides (e.g., 10-200, 50-150,10-50, 20-100, or 20-50 nucleotides).

TABLE 1 Alkyne an N-hydroxysuccinimide ester of: i) but-3-ynoic acid ii)pent-4-ynoic acid iii) hex-5-ynoic acid iv) hept-6-ynoic acid Azide anN-hydroxysuccinimide ester of: i) 2-azidoacetic acid ii)3-azidopropionic acid iii) 4-azidobutyric acid iv) 4-azidopentanoic acidv) 5-azidohexanoic acid vi) 5-azido-2-nitrobenzoic acid Alkene anN-hydroxysuccinimide ester of: i) but-3-enoic acid ii) pent-4-enoic acidiii) hex-5-enoic acid iv) hept-6-enoic acid Tetrazine AnN-hydroxysuccinimide ester of: i)2-(4-(1,2,4,5-tetrazin-3-yl)phenyl)acetic acid ii)2-(4-(1,2,4,5-tetrazin-3-yl)benzylamino)acetic acid iii)4-(4-(1,2,4,5-tetrazin-3-yl)benzylamino)-4-oxobutanoic acid iv)2-(6-(pyridin-2-yl)-1,2,4,5-tetrazin-3-yl)methyl- amino)butanoic acid v)2-((6-(pyridin-2-yl)-1,2,4,5-tetrazin-3-yl)methyl- amino)acetic acid vi)4-oxo-4-((6-(pyridin-2-yl)-1,2,4,5-tetrazin-3-yl)methyl- amino) butanoicacid α-halo- an N-hydroxysuccinimide ester of: benzyl i)(bromomethyl)benzoic acid ii) (chloromethyl)benzoic acid α-halo- anN-hydroxysuccinimide ester of: carbonyl i) bromo-acetic acid ii)chloroacetic acid Photo- an N-hydrosuccinimide ester of: crosslinker i)4-(7-oxo-7H-furo[3,2-g]chromen-9-yloxy)butanoic acid ii)4-azido-2,3,5,6-tetrafluorobenzoic acid iii)4-(3-(trifluoromethyl)-3H-diazirin-3-yl)benzoic acid iv)4-azido-2-hydroxybenzoic acid v) 3-benzoylbenzoic acid vi) 2-azidoaceticacid vii)5-azido-2-nitrobenzoic acid

The 3′ substituent of the functionalized RNA can also include a pausemoiety that is positioned between the linking moiety and the mRNA. Apause moiety is a moiety that causes the ribosome to stall or pausetranslation, thereby facilitating linkage of the nascent polypeptide tothe linking moiety of the functionalized RNA. When a purifiedtranslation system is used to translate the mRNA part of thefunctionalized RNA, a pause moiety may not be necessary to stalltranslation, for example, when release factors are omitted from thetranslation reaction. A pause moiety is generally useful for stallingtranslation when translation systems based on crude cell lysates areused. Pause moieties are known in the art. For example, a segment of anucleic acid other than RNA (e.g., DNA) or another polymer, for examplelocked nucleic acid (LNA), threose nucleic acid (TNA), glycerol nucleicacid (GNA), peptide nucleic acid (PNA), polyethylene glycol (PEG), or apeptide can be ligated to the RNA and employed as a pause moiety. Avariety of approaches for attaching such pause moieties to mRNA havebeen described (see U.S. Pat. Nos. 6,258,558; 6,416,950; and 6,429,300,all incorporated herein by reference). Other types of pause moieties canbe used. Certain RNA secondary structures that promote some level ofribosome stalling in natural and artificial genes, and thus play animportant role in the regulation of gene expression (e.g., hairpins andpseudoknots), can promote highly effective stalling. A variety ofhairpin and pseudoknot structures described in the literature can beinserted near the 3′ end of a coding RNA to effect stalling (Tate, W. P.and Brown, C. M. (1992) Biochemistry 31:2443-2450; Young, J. C. andAndrews, D. W. (1996) EMBO J. 15:172-181; Kozak, M. (2001) Nucl. AcidsRes. 29:5226-5232; Kontos, H., Napthine, S., and Brierley, I. (2001)Mol. Cell. Biol. 21:8657-8670; Plant, E. P. and Dinman, J. D. (2005)Nucl. Acids Res. 33:1825-1833; Yanofsky, C. (2007) RNA 13:1141-1154, andreferences cited therein, all of which are incorporated herein byreference).

The functionalized RNA described herein can be used to link apolypeptide encoded by the mRNA of the functionalized RNA to the mRNAthrough the 3′ substituent, and thereby generate an mRNA-polypeptidecomplex. The mRNA of the functionalized RNA is translated to produce apolypeptide using an in vitro translation system that includes anaminoacyl-tRNA comprising the linking amino acid. The linking amino acidhas a side chain that contains a member of a reactive pair that cancrosslink with the member of the reactive pair in the linking moiety.Non-limiting linking amino acids that can be used in these methodsinclude the D or L, D- or L-N-methyl, and D- or L-N-alkyl versions of:(a) β-azidoalanine; (b) azidohomoalanine; (c) azidonorvaline; (d)4-ethynyl-phenylalanine; (e) 2-amino-hex-5-ynoic acid; (f)trans-4,5-dehydro-lysine; (g) cysteine; (h) lysine; (i) allylglycine;(j) propargylglycine; (k) 2,3-diaminopropionic acid; (l) vinylglycine;(m) p-azido-phenylalanine; (n) S-allyl-cysteine; (o)S-(2-aminoethyl)-cysteine; (p) ornithine; (q)2-amino-3-cycloallylpropanoic acid; (r)2-amino-2-(trans-cyclooct-4-enyl) acetic acid; and (s)2-amino-2-(trans-cyclooct-3-enyl) acetic acid.

Crosslinking between members of a reactive pair occurs when the linkingmoiety occupies the A site of the ribosome. In some cases, thecrosslinking step requires the addition of an agent or a treatment. Forexample, copper ion can be added to promote crosslinking between analkyne and an azide. The translation system can be treated with lightand ethanethiol or β-mercaptoethanol to activate crosslinking between analkene and a thiol or an amine. Aqueous iodine or bromine can also beused to promote crosslinking between an alkene and a thiol. When aphotocrosslinker is involved, light, e.g., UV light, is used to activatethe crosslinking reaction. The agent or treatment is added at a timewhen the ribosome has completed translation of the functionalized RNAand has stalled, and the functionalized RNA and ribosome have beenincubated for a sufficient amount of time to permit the linking moietyof the 3′ substituent to occupy the A site of the ribosome. It can takeanywhere between 30 minutes to 24 hours or more after startingtranslation to provide sufficient time for the linking moiety to occupythe A site. A high potassium and magnesium wash step could be carriedout to improve occupancy of the A site by the linking moiety. Theoptimal interval for allowing the in vitro translation reaction toproceed before adding the agent or treatment to promote crosslinking canbe determined by assaying the yield of crosslinked mRNA-polypeptidecomplexes, e.g., by electrophoresis.

The methods described herein generate novel mRNA-polypeptide complexes.The mRNA-polypeptide complexes may include any non-RNA components of thefunctionalized RNAs used to generate the mRNA-polypeptide complexes. Thefunctionalized RNAs and the polypeptides in the complexes are linked viavarious bridging groups, depending on what reactive pairs are involved.For example, the mRNA and polypeptide may be linked through a bridginggroup selected from the group of: a triazole, a thioether, a secondaryamine, a pyridazine, a 3,4-diazanorcaradiene, benzylthioether, and abenzylamine. A “bridging group” as used herein is the residue formed bythe reaction between the two members of a reactive pair. Table 2 is anon-exhaustive list of the various types of bridging groups createdbetween the functionalized RNA and the polypeptide by using differentreactive pairs. Also see FIGS. 9A-C for examples of the bridging groupscreated between a functionalized RNA and a polypeptide using the methodsdescribed herein.

Provided herein are libraries of any of the functionalized RNAs and themRNA-polypeptide complexes described herein. In some embodiments ofthese libraries, the mRNA in each complex encodes a different protein oreach mRNA in each mRNA-polypeptide complex encodes a different protein.Some embodiments of these libraries contain a plurality of differentmRNA-polypeptide complexes. As described in detail below, libraries offunctionalized RNAs may be used to generate a library ofmRNA-polypeptide complexes, that may in turn be used to screen forpolypeptides that interact with (e.g., specifically bind) a target.

Also provided are translation systems that contain any of the librariesof functionalized RNAs described herein and at least one aminoacylatedtRNA containing any of the linking amino acids described herein. In someembodiments of these translation systems, the aminoacylated tRNA is anaminoacylated suppressor tRNA.

TABLE 2 Bridging Groups Formed Between Reactive Pair Functionalized RNAand Polypeptide an azide and an alkyne Triazole an alkene and a thiolThioether an alkene and an amine secondary amine a tetrazine and atrans-cyclooctane Pyridazine a tetrazine and a cyclopropene3,4-diazanorcaradiene a tetrazine and a Pyridazinebicyclo[2.2.1]hept-2-ene a tetrazine and a norbornene Pyridazine anα-halo-benzyl and a thiol Benzylthioether an α-halo-benzyl and an amineBenzylamine an α-halo-carbonyl and a thiol Thioether an α-halo-carbonyland an amine secondary amineMethods of Making and Using Libraries

The methods described herein for linking an mRNA to a polypeptideencoded by the mRNA through a linking amino acid and a linking moietyand for linking an mRNA to a polypeptide encoded by the mRNA through alinking tRNA can be used to create libraries of polypeptides and toselect novel polypeptides that have specific target-binding or otheractivities. Accordingly, provided herein are methods of selecting for apolypeptide (or an mRNA encoding a polypeptide) that interacts with atarget or exhibits another desired, specific activity. Also providedherein are methods of using libraries of the mRNA-polypeptide and themRNA-tRNA-polypeptide complexes described herein to optimize the bindingor functional properties of a polypeptide. A library will generallycontain at least 10² members, more preferably at least 10⁶ members, andmore preferably at least 10⁹ members (e.g., any of the mRNA-polypeptidecomplexes and/or mRNA-tRNA-polypeptide complexes described herein). Insome embodiments, the library will include at least 10¹² members or atleast 10¹⁴ members. In general, the members will differ from each other;however, it is expected there will be some degree of redundancy in anylibrary. The library can exist as a single mixture of all members, orcan be divided into several pools held in separate containers or wells,each containing a subset of the library, or the library can be acollection of containers or wells on a plate, each container or wellcontaining just one or a few members of the library.

A library of mRNAs, each mRNA comprising a member of a reactive pairthat can participate in crosslinking (often referred to as afunctionalized RNA) to an appropriately modified polypeptide or tRNA.Each mRNA in the library preferably comprises a translation initiationsequence, a start codon, and a variable polypeptide (e.g., protein orshort peptide) coding region that is generated by, for example, a randomor semi-random assembly of nucleotides, and varies from mRNA to mRNA inthe library (though there will likely be some degree of redundancywithin the library). The translation initiation sequence, start codon,and variable polypeptide coding region can be flanked by known, fixedsequences that can be used for PCR amplification of the mRNA, e.g.,after selection. Other fixed sequences that can be present include thosecorresponding to restriction enzyme recognition sequences as well assequences that encode amino acids that can participate in chemical orenzymatic cross-linking reactions, such that the polypeptide producedcan be modified or derivatized after translation, or that encode a fixedC-terminal extension.

Once a library of functionalized RNAs of the invention is generated, themRNAs present in the members of the library can be translated. Theresulting polypeptides (e.g., displayed polypeptides) will be linked totheir corresponding functionalized RNAs as described herein (e.g., as anmRNA-polypeptide complex or an mRNA-tRNA-polypeptide complex).Translation is carried out using a translation system containing a setof aminoacyl-tRNAs that includes the appropriate linking tRNA or linkingamino acid matched to the linking moiety utilized in the functionalizedRNAs in the library. Aminoacyl-tRNAs containing linking amino acids,other unnatural amino acids, or natural amino acids can be generatedusing methods known in the art and described herein. When employing alinking amino acid, the linking amino acid can be attached to the tRNAvia an ester bond or a stable, non-hydrolyzable covalent bond (e.g., anamide bond) (see Fraser, T. H. and Rich, A., (1973) Proc. Natl. Acad.Sci. USA 70:2671-2675; Merryman, C. et al. (2002) Chemistry and Biology9:741-746; U.S. Pat. No. 6,962,781). Other aminoacyl-tRNAs that may beused include those with the linking amino acids attached via a stable 3′hydrazide, oxyamide, methylene, or oxymethylene linkage. When employinga linking tRNA, the amino acid attached to the linking tRNA ispreferably attached via a stable, non-hydrolyzable covalent bond.

Numerous in vitro translation systems have been described in theliterature. The most common systems utilize rabbit reticulocyte lysates,wheat germ extracts, or E. coli extracts, which are available from anumber of commercial sources in kit form (e.g., Ambion, Austin, Tex.;Promega, Madison, Wis.; Novagen/EMD Chemicals, Gibbstown, N.J.; Qiagen,Valencia, Calif.). Other systems based on purified translation factorsand ribosomes have been described (Shimizu, Y. et al. (2001) Nat.Biotech. 19:751-755; Josephson, K., Hartman, M. C. T., and Szostak, J.W. (2005) J. Am. Chem. Soc. 127: 11727-11735; Forster, A. C. et al.(2003) Proc. Natl. Acad. Sci. USA 100: 6353-6357). A system based onpurified E. coli translation factors is commercially available(PURExpress™; New England Biolabs, Ipswich, Mass.). In general, theconditions recommended by the kit manufacturers are suitable for invitro translation of the mRNA portion of the functionalized RNAsdescribed herein. The optimal interval for allowing the in vitrotranslation reaction to proceed can be determined by assaying the yieldof crosslinked mRNA-polypeptide complexes or mRNA-tRNA-polypeptidecomplexes, e.g., by electrophoresis.

As described above, the crosslinking reaction between members of thereactive pair occurs after translation has been completed and theribosome has stalled, and the linking moiety has occupied the A site ofthe ribosome. In some embodiments, one or more additional agents ortreatments are required, e.g., after translation, to inducecrosslinking. The aminoacyl-tRNA containing the linking tRNA or thelinking amino acid may recognize a sense codon or a non-sense (stop)codon. In some embodiments, the linking tRNA or the tRNA attached to thelinking amino acid recognizes a sense codon that immediately precedesone or two stop codons on the functionalized RNA. In some embodiments,the tRNA recognizes a stop codon (i.e., is a suppressor tRNA) on thefunctionalized RNA. It may be advantageous to use a purified in vitrotranslation system lacking release factors (e.g., Tan, Z. et al. (2005).Methods 36:279-290; and U.S. Pat. No. 6,977,150). The absence of releasefactors will prevent premature dissociation of the peptidyl tRNA fromthe ribosome. The absence of release factors is especially useful ifcrosslinking depends on the addition of an agent or the use ofultraviolet light to initiate the crosslinking reaction.

Translation can be performed with naturally-occurring amino acids (i.e.,the 20 natural proteinogenic amino acids commonly found in naturalproteins). The 20 natural proteinogenic amino acids are alanine,arginine, asparagine, aspartic acid, cysteine, glutamic acid, glutamine,glycine, histidine, isoleucine, leucine, lysine, methionine,phenylalanine, proline, serine, threonine, tryptophan, tyrosine, andvaline. Alternatively, “unnatural” amino acids, which have side chainsnot present in the 20 naturally-occurring amino acids listed above, canbe used. Unnatural amino acids include, but are not limited to:S-(2-aminoethyl)-L-cysteine, 4-fluoro-L-tryptophan,L-β-azidohomoalanine, β-hydroxy-L-norvaline, 4,4-difluoro-L-glutamate,5′5′5′-trifluoro-L-leucine, L-2-amino-hex-5-ynoic acid, L-canavanine,3-fluoro-L-valine, 2-fluoro-L-phenylalanine, 4-fluoro-L-phenylalanine,4-chloro-L-phenylalanine, O-methyl-L-tyrosine, 4-pyridinepropanioc acid,2-amino-2-(1H-tetrazol-5-yl) acetic acid, 5-fluoro-L-tryptophan,L-t-butyl glycine, 4-fluoro-L-glutamate, 7-aza-L-tryptophan,trans-4,5-dehydro-L-lysine, O-methyl-L-threonine, L-norleucine,2-amino-3-methoxybutanoic acid, L-ethionine, L-glutamic acid-γ-methylester, 3,4-dehydro-L-proline, L-crotylglycine,1-aminocyclopentanecarboxylic acid, L-threo-β-hydroxy aspartic acid,1-aminocyclohexane-1-carboxylic acid, quisqualic acid,4-thia-L-isoleucine, β-t-butyl-L-alanine, 3-fluoro-L-tyrosine,thiazolidine-2-carboxylic acid, α-methyl-L-aspartic acid, L-norvaline,α-methyl-L-proline, L-thiazolidine-4-carboxylic acid,L-azetidine-2-carboxylic acid, β-methyl-L-phenylalanine,5-hydroxy-L-tryptophan, 3-(thianaphthen-3-yl)-L-alanine, ibotenic acid,4-methyl-L-glutamate, 4-aza-L-leucine, 3-(2-thienyl)-L-alanine,L-β-(1,2,4-triazol-3-yl-alanine, L-phenylglycine, L-allylglycine,p-nitro-L-phenylalanine, L-p-iodo-phenylalanine, N-methyl-L-aspartate,N-methyl-L-leucine, alpha-hydroxy amino acids, N-methyl amino acids,N-alkyl amino acids, alpha-alkyl amino acids, beta-amino acids, D-aminoacids, and other unnatural amino acids known in the art. (See, e.g.,Josephson et al., (2005) J. Am. Chem. Soc. 127: 11727-11735; Forster, A.C. et al. (2003) Proc. Natl. Acad. Sci. USA 100: 6353-6357; Subtelny etal., (2008) J. Am. Chem. Soc. 130: 6131-6136; Hartman, M. C. T. et al.(2007) PLoS ONE 2:e972; and Hartman et al., (2006) Proc. Natl. Acad.Sci. USA 103:4356-4361). Essentially any amino acid that, when attachedto an appropriate tRNA, can be assembled into a polymer by natural ormutant ribosomes can be used (see Sando, S. et al., (2007) J. Am. Chem.Soc. 129:6180-6186; Dedkova, L. et al. (2003)J. Am. Chem. Soc. 125:6616-6617; Josephson, K., Hartman, M. C. T., and Szostak, J. W. (2005)J. Am. Chem. Soc. 127:11727-11735; Forster, A. C. et al. (2003) Proc.Natl. Acad. Sci. USA 100:6353-6357; Subtelny, A. O., Hartman, M. C. T.,and Szostak, J. W. (2008) J. Am. Chem. Soc. 130:6131-6136; and Hartman,M. C. T. et al. (2007) PLoS ONE 2:e972).

When unnatural amino acids are desired, it may be advantageous to use apurified translation system that lacks endogenous aminoacylated tRNAs(Shimizu, Y. et al. (2001) Nat. Biotech. 19:751-755; Josephson, K.,Hartman, M. C. T., and Szostak, J. W. (2005) J. Am. Chem. Soc. 127:11727-11735; Forster, A. C. et al. (2003) Proc. Natl. Acad. Sci. USA100: 6353-6357). If unnatural amino acids are used with an in vitrotranslation system based on a lysate or extract, it may be desirable todeplete the extract of endogenous tRNAs, as previously described (seeJackson, R. J., Napthine, S., and Brierley, I. (2001) RNA 7:765-773).

When using natural amino acids with an in vitro translation system basedon a lysate or extract, translation is dependent on the enzymaticcharging of amino acids onto tRNAs by tRNA synthetases, all of which arecomponents of the extracts. Alternatively, in vitro translation systemsthat use purified translation factors and ribosomes, or tRNA-depletedextracts, require that aminoacylated tRNAs be provided. In theseinstances, purified or in vitro synthesized tRNAs can be charged withamino acids using chemical (see Frankel, A., Millward, S. W., andRoberts, R. W. (2003) Chem. Biol. 10:1043-1050) or enzymatic procedures(Josephson, K., Hartman, M. C. T., and Szostak, J. W. (2005) J. Am.Chem. Soc. 127: 11727-11735; Murakami, H. et al. (2006) Nat. Methods3:357-359).

Numerous publications describe the recovery of mRNA-displayedpolypeptides from translation complexes, and these are suitable for usewith the methods described herein (Liu, R. et al. (2000). MethodsEnzymol. 318:268-293; Baggio, R. et al. (2002). J. Mol. Recognit.15:126-134; U.S. Pat. No. 6,261,804). The recovery of mRNA-displayedpolypeptides may be facilitated by the use of various “tags” that areincluded in the polypeptide by translation of fixed sequences of thepolypeptide coding sequence and which bind to specific substrates ormolecules. Numerous reagents for capturing such tags are commerciallyavailable, including reagents for capturing the His-tag, FLAG-tag,glutathione-S-transferase (GST) tag, strep-tag, HSV-tag, T7-tag, S-tag,DsbA-tag, DsbC-tag, Nus-tag, myc-tag, hemagglutinin (HA)-tag, or Trx-tag(Novagen, Gibbstown, N.J.; Pierce, Rockford, Ill.). mRNA-displayedpeptides can also be isolated by binding of a polyA tail on the mRNA topolydT resin, or a combination of a polyA tail and a His-tag. modifiedto improve or alter their properties. One way to accomplish this is byincorporating unnatural amino acids with reactive side chains into thepolypeptides that make up the library. After translation, the newlyformed polypeptides can be reacted with molecules that reactspecifically with the reactive side chain of the incorporated aminoacid. For example, an amino acid with a terminal alkyne side chain canbe incorporated into the polypeptide library and subsequently reactedwith an azido sugar, creating a library of displayed polypeptides withsugars attached at the positions of the alkynyl side chains (Josephson,K., Hartman, M. C. T., and Szostak, J. W. (2005) J. Am. Chem. Soc. 127:11727-11735). A variety of reactive side chains can be used for suchpost-translational conjugation, including amines, carboxyl groups,azides, terminal alkynes, alkenes, and thiols.

One particularly useful modification is based on the cross-linking ofamino acids to produce cyclic structures. Cyclic regions in a proteincontain a rigid domain which reduces conformational flexibility anddegrees of rotational freedom, leading to very high affinity binding totarget proteins. A number of methods for cyclizing a polypeptide areavailable to those skilled in the art and are incorporated herein byreference. Typically, the chemical reactivity of specific amino acidside chains and/or the carboxyl or amino termini of the polypeptide areexploited to crosslink two sites of the polypeptide to produce a cyclicmolecule. In one method, the thiol groups of two cysteine residues arecross-linked by reaction with dibromoxylene (see Timmerman, P. et al.,(2005) ChemBioChem 6:821-824). Tri- and tetrabromoxylene can be used toproduce polypeptides with two and three loops, respectively. In anotherexemplary method, a side chain amino group and a terminal amino groupare cross-linked with disuccinimidyl glutarate (see Millward, S. W. etal., J. Am. Chem. Soc. 127:14142-14143, 2005). In other approaches,cyclization is accomplished by making a thioether bridging group betweentwo sites on the polypeptide. One chemical method relies on theincorporation of an N-chloroacetyl modified amino acid at the N-terminusof the polypeptide, followed by spontaneous reaction with the thiol sidechain of an internal cysteine residue (see Goto, Y. et al. (2008) ACSChem. Biol. 3:120-129). An enzymatic method relies on the reactionbetween (1) a cysteine and (2) a dehydroalanine or dehydrobutyrinegroup, catalyzed by a lantibiotic synthetase, to create the thioetherbridging group (see Levengood, M. R. and Van der Donk, W. A., Bioorg.and Med. Chem. Lett. 18:3025-3028, 2008).

After the in vitro translation reaction has been performed, and prior tothe selection step, the mRNA portion of the functionalized RNA istypically reversed-transcribed to produce a RNA-DNA hybrid molecule(i.e., a cDNA). This serves to protect the RNA from degradation, andalso prevents the RNA from folding into a secondary structure that couldbind to the selection target, which would lead to selection ofinappropriate products (i.e., the selection of RNA aptamers rather thanpolypeptide aptamers).

A library of mRNA-polypeptide complexes or mRNA-tRNA-polypeptidecomplexes (also referred to herein as an mRNA display library) generatedusing the above described methods, and which may or not have beensubjected to a post-translational modification (such as cyclization ofthe polypeptide, as described above), can be subjected to a batchselection step to isolate those complexes displaying desirablepolypeptides. A target used in the selection step is typically isolatedby purification from a natural biological source or from a recombinantDNA expression system. Alternatively, the target may be used in anon-purified state or may be prepared by chemical synthesis. The targetmay be a protein, such as a cell-surface receptor (for example, acytokine or neurotransmitter receptor), an enzyme, a transcriptionfactor, a hormone, a cytokine, an antibody, an antibody domain, an ionchannel, a chaperone, an adhesion molecule, or any other nuclear,cytoplasmic, cell-surface, or serum protein, or a fragment of such aprotein. The target may also be a lipoprotein, polysaccharide,glycoprotein, proteoglycan, peptidoglycan, lipid, small molecule, RNA,DNA, or any other nucleic acid molecule. The target may be any substanceor structure for which it is desirable to isolate a binding polypeptide.

Typically, a purified target (e.g., a protein or any of the targetmolecules described herein) is conjugated to a solid substrate, such asan agarose or synthetic polymer bead. The conjugated beads are mixedwith the mRNA display library and incubated under conditions (e.g.,temperature, ionic strength, divalent cations, and competing bindingmolecules) that permit specific members of the library to bind thetarget. Alternatively, the purified target protein can be free insolution and, after binding to an appropriate polypeptide, themRNA-polypeptide complex or the mRNA-tRNA-polypeptide complex with abound target is captured by an antibody that recognizes the target(e.g., target protein) at a site distinct from the site where thedisplayed polypeptide binds. The antibody itself can be bound to a bead,or it may be subsequently captured by a suitable substrate, such asProtein A or Protein G resins. The binding conditions can be varied inorder to change the stringency of the selection. For example, lowconcentrations of a competitive binding agent can be added to ensurethat the selected polypeptides have a relatively higher affinity.Alternatively, the incubation period can be chosen to be very brief,such that only polypeptides with high k_(on) rates will be isolated. Inthis manner, the incubation conditions play an important role indetermining the properties of the selected polypeptides. Negativeselections can also be employed. In this case, a selection to removepolypeptides with affinity to the substrate to which the target is bound(e.g., Sepharose) is carried out by applying the displayed library tosubstrate beads lacking the target protein. This step can remove mRNAsand their encoded polypeptides that are not specific for the targetprotein. Numerous references describing how to conduct selectionexperiments are available. (See, e.g., U.S. Pat. No. 6,258,558; Smith,G. P. and Petrenko, V. A., (1997) Chem. Rev. 97:391-410; Keefe, A. D.and Szostak, J. W. (2001) Nature 15:715-718; Baggio, R. et al. (2002) J.Mol. Recog. 15:126-134; Sergeeva, A. et al. (2006) Adv. Drug Deliv. Rev.58:1622-1654).

The frequency at which binding molecules are present in a library ofrandom sequences is expected to be very low. Thus, in the initialselection step, very few polypeptides meeting the selection criteria(and their associated mRNAs) are expected to be recovered. Typically,the selection is repeated with mRNAs selected from the first round ofselection. This is accomplished by using PCR to amplify the mRNAs orcorresponding cDNAs selected in the first round, followed by in vitrotranscription to produce a new library of mRNAs. PCR primerscorresponding to the 5′ and 3′ ends of the mRNAs in the library areused. Typically, the 5′ primer will extend in the 5′ direction beyondthe end of the mRNA so that a bacterial promoter, such as a T7 promoter,is added to the 5′ end of each amplified molecule. Once amplified, thedouble-stranded DNA can be used in an in vitro transcription reaction togenerate the mRNA for a second round of selection. This mRNA is modifiedas necessary, e.g., by incorporating it into functionalized RNA asdescribed above.

The selection process typically involves a number of rounds or cycles,in which the pool of selected molecules is incrementally enriched in aspecific set of sequences at the end of each round. The selectionconditions may be the same for each round, or the conditions may change,for example, in order to increase the stringency of selection in laterrounds. The progress of selection may be monitored by the use ofisotopically-labeled amino acids, such as ³⁵S methionine. The amount ofradiolabeled polypeptide bound to the target at each round is measured,and a progressive increase in recovered radiolabel is indicative of aprogressive enrichment in RNA molecules encoding polypeptides withbinding affinity to the target. After any round, the PCR products may becloned and sequenced. Generally, cloning and sequencing is performedafter a round in which appreciable (>5%) amounts of radiolabeledpolypeptide are recovered in the target-bound pool. Sequences that arefound in multiple isolates are candidates for encoding polypeptides thatbind specifically to the target. Alternatively, high throughputsequencing of thousands of clones can be performed after early rounds,such as after the third and fourth round. Sequences that increase infrequency between the third and fourth rounds are candidates forencoding polypeptides that bind specifically to the target. Thepolypeptide encoded by any sequence may be expressed or synthesized andtested for binding affinity to the original target protein used in theselection.

The libraries and methods of the present invention may be used tooptimize the function or properties of a polypeptide. In one approach,mutagenic PCR (Keefe, A. D. and Szostak, J. W. (2001). Nature15:715-718) is used to introduce sequence variation in the library oncethe population is enriched in polypeptides with a certain level ofbinding affinity. Alternatively, a single RNA sequence encoding apolypeptide with defined binding properties can be replicated but with adefined level of mutations, or mutagenic PCR can be performed to producea pool of mutant molecules. The resulting mixture of mRNA moleculesproduced from such a pool is expected to encode polypeptides with arange of improved, similar, or reduced affinities as compared to thestarting sequence, and a selection performed on mRNAs from such a poolmay be expected to identify polypeptides with improved affinity if anappropriate stringency regimen is used during the selection.

In a second approach, optimization is performed in a directed manner. Asequence encoding a polypeptide with established binding or functionalproperties is subjected to site-directed mutagenesis, whereby a seriesof sequences is produced, with each sequence having one codon replacedwith, for example, an alanine codon. The number of sequences in the setis equal to the number of amino acid residues that are to be mutated.The polypeptide product of each “alanine scanning” mutant is tested forbinding or functional properties. Sites at which an alanine substitutionaffects the binding or function of the polypeptide are consideredcritical residues. Alternatively, the sequences can be pooled, subjectedto one or more rounds of a high stringency selection, and a pool ofsequences representing high affinity binding polypeptides is isolated.Critical residues are identified as those that cannot be substituted byan alanine residue without loss of activity. Once the critical residuesare identified, a pool of mRNA molecules encoding a wide variety ofnatural (or unnatural) amino acids at each critical position isproduced. The resulting pool is subjected to one or more rounds of ahigh stringency selection (with the appropriate mixture of tRNAs chargedwith natural or unnatural amino acids), and sequences representing highaffinity binding polypeptides are isolated. In this manner, an optimalpolypeptide can be identified. Since the optimal sequence may notnecessarily be identified by combining optimal residues at individualsites, it is useful to test mutations at multiple sites in combination.

EXAMPLES Example 1 Synthesis of Bifunctional CrosslinkerN,N′-bis-bromoacetyl-1,2-diaminobenzene

1,2-phenylenediamine (2.75 g, 25 mmoles) was dissolved in 100 mL of drytetrahydrofuran (THF) containing triethylamine (8.71 mL, 62.5 mmoles)and cooled in an ice bath. A solution of bromoacetylbromide in THF (50mmoles, 4.35 mL in 15 mL THF) was added slowly and the resultingsolution is stirred at 0° C. for 1 hour followed by an additional 3hours at room temperature. Water (25 mL) was then added and the solutionallowed to stand until a precipitate formed. The precipitate comprisingN,N′-bis-bromoacetyl-1,2-diaminobenzene was filtered off andre-crystallized from hot methanol.

Donor oligonucleotide containing a ^(4S)U residue was purchased asTOM-protected RNA from the Keck oligonucleotide facility at YaleUniversity (New Haven, Conn.). The lyophilized powder was dissolved in0.1 mL of DMSO. Once fully dissolved, triethylamine trihydrofluoride(0.125 mL; neat) was added and the solution incubated at 65° C. for 2hours. The deprotected RNA was precipitated with butanol, washed oncewith absolute cold ethanol and once with 70% ethanol/water.

The pellet was dissolved in 0.1 mL of diethylpyrocarbonate(DEPC)-treated water and 10 μL were purified by ion exchange HPLC on aDionex DNA-pac column using a gradient of 0.01 M NaCl to 1 M NaClcontaining EDTA in nuclease free water. The major fraction was collectedand desalted in an OPC purification cartridge (ABI; Carlsbad, Calif.),using the conditions recommended by the manufacturer. 1.5 nmole of thedonor oligonucleotide was obtained and HPLC with high resolution massspectroscopy was used to confirm the presence of the thiouridineresidue.

In order to activate the ^(4S)U oligonucleotide for crosslinking, 1.5nmoles of donor oligonucleotide (25 μL of a 83 μM solution) wasdissolved in 70 μL of 0.1 M K₂HPO₄/KH₂PO₄ pH: 8.0. A solution of theelectrophile N,N′-bis-bromoacetyl-1,2-diaminobenzene was prepared (1 mgin 30 μL of dimethylformamide (DMF)) and added to the ^(4S)Uoligonucleotide. The sample was allowed to sit at room temperature for1.5 hours and the reaction terminated by passing the sample through amicro-biospin 6 column (Bio-Rad; Hercules, Calif.) and precipitated with1 mL of butanol. The oligo was washed once with ethanol, centrifugated,and evaporated to dryness on a Speed-Vac™ lyophilizer.

The activated oligonucleotide is ligated onto the end of an mRNApreparation containing the sequences of interest by splint ligation, inwhich the mRNA and activated oligonucleotide are annealed to a DNAoligonucleotide containing sequences complementary to both the mRNA andthe activated oligonucleotide and are joined together using T4 DNAligase (Das, S. R. and Piccirilli, J. A., Nat. Chem. Biol. 1:45-52,2005). The resulting mRNA is then subjected to in vitro translation andtRNA crosslinking.

Example 2 Preparation of Furan Modified Oligonucleotides from 2′-aminoUridine (or Cytidine) Containing Oligonucleotides

Oligonucleotides containing a single 2′-amino nucleoside moiety aresynthesized via solid phase synthesis using TOM-protected RNA amidites(Glen Research; Sterling, Va.) and 2′-amino uridine CED phosphoramidite(Chemgenes; Wilmington, Mass.) on an Expedite™ (Millipore; Billerica,Mass.) RNA synthesizer using the conditions recommended by themanufacturers.

2-furyl propionic acid is synthesized as described by Halila et al.(Chem. Commun. 21:936-938, 2005) from commercially available3-(2-furyl)acrylic acid. 2-furyl propionic acid (2 g, 14.3 mmoles) isdissolved in anhydrous tetrahydrofuran (50 mL) followed by addition oftriethylamine (2 molar equivalents) and cooled to 0° C.N-hydroxysuccinimide (1.1 molar equivalent) is then added followed bysolid dicyclohexylcarbodiimide (1.1 molar equivalent), which is added inportions during a period of 30 minutes. Once the addition is completethe reaction is allowed to reach room temperature and stirred overnight.The volatiles ware evaporated and the reaction diluted withdichloromethane (10 mL). The product is passed through a short columnpacked with silica gel and eluted with dichloromethane. The solvent isevaporated to obtain the desired compound.

The crude oligonucleotide (250 μmoles) containing 2′-amino uridine isdissolved in 100 μL of 70 mM boric acid (pH 8.5) and cooled on anice-bath. Formamide (60 μL) is added, followed by addition of a freshlyprepared solution of NHS-furan (15 molar equivalents) in DMF (40 μL)(Handbook of RNA Biochemistry; Hartmann, R. K., Biendereif, A., Schon,A., and Westhof, E., 2005). A similar procedure can be used forisocyanate- and isothiocyanate-activated furan derivatives.

The resulting solution is incubated for 60 minutes on ice and a secondaliquot of NHS-furan ester is added and incubated for an additionalhour. The solution is extracted twice with chloroform (300 μL) at roomtemperature and precipitated by addition of 20 μL of 3 M sodium acetateand 1.3 mL ethanol. The mixture is cooled to −70° C. for 1 hour and theprecipitated oligonucleotide pelleted by centrifugation. Theoligonucleotide is washed once with 70% ethanol, centrifuged, andevaporated to dryness using a Speed-Vac™ lyophilizer. Theoligonucleotide is purified by preparative reverse phase HPLC on a C-18column using a linear gradient of triethylammonium acetate (20 mM) to60% acetonitrile in triethylammonium acetate (20 mM). Fractionscontaining the oligonucleotide are lyophilized to dryness.

The furan-modified oligonucleotide is ligated onto the end of an mRNApreparation containing the sequences of interest by splint ligation, inwhich the mRNA and activated oligonucleotide are annealed to a DNAoligonucleotide containing sequences complementary to both the mRNA andthe activated oligonucleotide and are joined together using T4 DNAligase (New England BioLabs, Ipswich, Mass.). The resulting mRNA is thensubjected to in vitro translation and tRNA crosslinking.

Example 3 Preparation of Furan-modified Oligonucleotides from 2′-aminoFuran Modified Phosphoramidite

2′-amino furan-modified phosphramidite[2′-deoxy-2′-(2-furyl-2-ethoxycarbonylamino)-5′-O-(4,4′-dimethoxytrityl)uridine3′-(2-cyanoethyl N,N-diisopropylphosphoramidite)] is prepared from its2′-amino nucleoside as described previously (Halila et al., 2005,supra). RNA oligonucleotides containing the 2′-amino furan moiety aresynthesized via solid phase synthesis using TOM-protected RNAphorphoramidites (Glen Research; Sterling, Va.) and 2′-amino uridine CEDphosphoramidite (Chemgenes; Wilmington, Mass.) on an Expedite(Millipore; Billerica, Mass.) RNA synthesizer using the conditionsrecommended by the manufacturers. The desalted oligonucletide ispurified by denaturing PAGE electrophoresis (15% urea) and furtherpurified via RP-HPLC on a C-18 column using a linear gradient oftriethylammonium acetate (20 mM) to 60% acetonitrile in triethylammoniumacetate (20 mM). Fractions containing the desired oligonucleotide arelyophilized to dryness.

Example 4 Crosslinking of an mRNA to a tRNA Via a 4-oxo-2-PentenalMoiety

An mRNA containing a 2′-amino furan modification is incubated with an invitro translation reaction mixture at 37° C. After 30 min4-oxo-2-pentenal is formed by adding 1 molar equivalent (with respect tomRNA) of N-bromosuccinimide, freshly prepared in phosphate buffercontaining 10 mM NaCl, pH 7. The crosslinking reaction of the4-oxo-2-pentenal mRNA is allowed to proceed for 8 hours.

Example 5 Preparation of 2-amino-6 vinylpurine Phosphoramidite

The 2-amino-6-vinylpurine phosphoramidite2-acetamido-5′-O-DMT-2′-O-TOM-3′-cyanoethyldiisopropylphospharamidite-9-D-ribofuranosyl-6-(2-(phenylthio)ethyl)purine(shown as compound (1) in FIG. 6) is synthesized as described below.2-amino-9-(2,3,5-tri-O-tert-butyldimethylsilyl-D-ribofuranosyl)-6-vinylpurine(shown as compound (2) in FIG. 6) is synthesized as described byNagatsugi et al. (Nucleic Acids Symp. Ser. 67-68, 1997). 0.22 g (2mmoles) of thiophenol is added to a 40 mL solution of 1.27 g (2 mmoles)of2-amino-9-(2,3,5-tri-O-tert-butyldimethylsilyl-D-ribofuranosyl)-6-vinylpurinein 20% dichloromethane in ethanol and the resulting solution stirred atroom temperature for 1 hour. The product,2-amino-9-(2,3,5-tri-O-tert-butyldimethylsilyl-D-ribofuranosyl)-6-(2-(phenylthio)ethyl)purine(shown as compound (3) in FIG. 6), is purified by silica gelchromatography using chloroform/methanol (99:1).

The 2-amino group of2-amino-9-(2,3,5-tri-O-tert-butyldimethylsilyl-D-ribofuranosyl)-6-(2-(phenylthio)ethyl)purineis protected by amidation to produce2-acetamido-9-(2,3,5-tri-O-tert-butyldimethylsilyl-D-ribofuranosyl)-6-(2-(phenylthio)ethyl)purine(shown as compound (4) in FIG. 6). To produce compound (4) (in FIG. 6),a solution of 1.33 g (1.7 mmoles) of compound (3) (in FIG. 6) inpyridine (50 mL) is cooled to 0° C. and 1.5 molar equivalents acetylchloride is added. The solution is allowed to reach room temperature andstirred for 2 hours. Water (5 mL) is added to quench the unreactedacylating agent and the solution stirred for additional 10 minutes.Volatiles are evaporated under reduced pressure and the residuedissolved in chloroform (75 mL) and washed twice with a saturatedsolution of sodium bicarbonate (2×50 mL), water (50 mL), and brine (50mL). The organics are dried with magnesium sulfate, filtered, andevaporated under reduced pressure to yield compound (4) (in FIG. 6).

2-acetamido-9-D-ribofuranosyl-6-(2-(phenylthio)ethyl)purine (shown ascompound (5) in FIG. 6) is produced by removing thetert-butyldimethylsilyl (TBDMS) protecting groups of compound (4) (inFIG. 6). 1.4 g (1.77 mmoles) of compound (4) (in FIG. 6) is dissolved in10 mL THF, treated with 10 mL of a 1 M tetra-n-butylammonium fluoride(TBAF) solution in THF, and stirred at room temperature for 6 hours. Thevolatiles are evaporated and the product,2-acetamido-9-D-ribofuranosyl-6-(2-(phenylthio)ethyl)purine, is purifiedby silica gel chromatography using methanol:chloroform (8%).

A dimethoxytrityl (DMT) protecting group is added at the 5′ position ofthe ribose moiety to produce2-acetamido-5′-O-DMT-9-D-ribofuranosyl-6-(2-(phenylthio)ethyl)purine(shown as (6) in FIG. 6).2-acetamido-9-D-ribofuranosyl-6-(2-(phenylthio)ethyl)purine (compound(5) in FIG. 6; 0.75 g; 1.6 mmoles) is dissolved in anhydrous pyridine(16 mL) and treated with DMT-Cl (1.05 molar equivalents) with stirringat room temperature for 6 hours. Water (5 mL) is added and the solutionstirred for additional 10 minutes. Volatiles are evaporated underreduced pressure and the residue dissolved in dichloromethane (75 mL)and washed twice with a saturated solution of sodium bicarbonate (2×50mL), water (50 mL), and brine (50 mL). The organic residue is dried withmagnesium sulfate, filtered, and evaporated under reduced pressure toyield2-acetamido-5′-O-DMT-9-D-ribofuranosyl-6-(2-(phenylthio)ethyl)purine,which is used in the next step without purification.

2-acetamido-5′-O-DMT-9-D-ribofuranosyl-6-(2-(phenylthio)ethyl)purine isfurther modified by addition of a 2′ (triisopropylsilyl)oxy]methyl groupto produce2-acetamido-5′-O-DMT-2′-O-TOM-9-D-ribofuranosyl-6-(2-(phenylthio)ethyl)purine(shown as (7) in FIG. 6).2-acetamido-5′-O-DMT-9-D-ribofuranosyl-6-(2-(phenylthio)ethyl)purine(1.2 g, 1.6 mmoles) is dissolved in 1,2-dichloroethane (16 mL) andtreated with t-Bu₂SnCl₂ (1.0 molar equivalents) anddiisopropylethylamine (3.5 molar equivalents) and stirred at roomtemperature for 30 minutes. [(Triisopropylsilyl)oxy]methyl chloride (1.1molar equivalents) is added to the solution and stirred for additional30 minutes. Volatiles are evaporated under reduced pressure and theresidue dissolved in dichloromethane (75 mL), washed twice with asaturated solution of sodium bicarbonate (2×50 mL), and washed once withbrine (50 mL). The organics are dried with magnesium sulfate, filtered,and evaporated under reduced pressure.2-acetamido-5′-O-DMT-2′-O-TOM-9-D-ribofuranosyl-6-(2-(phenylthio)ethyl)purineis isolated by column chromatography using hexane/ethyl acetate 9:1→1:1.

2-acetamido-5′-O-DMT-2′-O-TOM-3′-cyanoethyldiisopropylphospharamidite-9-D-ribofuranosyl-6-(2-(phenylthio)ethyl)purine((1) in FIG. 6) is prepared by dissolving 0.6 g (0.6 mmoles) of2-acetamido-5′-O-DMT-3′-O-TOM-9-D-ribofuranosyl-6-(2-(phenylthio)ethyl)purinein dichloromethane (6 mL), cooling in an ice bath, and treating withdiisopropylethylamine (3 molar equivalents) and cyanoethyldiisopropyl-phosphoramidochloridite (1.2 molar equivalents). Thesolution is stirred for 2 hours at room temperature. Dichloromethane (40mL) is added and the solution is washed twice with 10% aqueous sodiumbicarbonate (2×30 mL) and water (30 mL). The organics are dried withmagnesium sulfate and the product is purified by column chromatographyon silica using hexane/ethyl acetate 9:1→1:1. The desired product isobtained as an off-white foam.

Example 6 Synthesis of Oligonucleotides Containing 2-amino-6-vinylpurine

RNA oligonucleotides containing a 2-amino-6-vinyl purine moiety aresynthesized via solid phase synthesis using TOM-protected RNA amidites(Glen Research; Sterling, Va.) and2-acetamido-5′-O-DMT-2′-O-TOM-3′-cyanoethyldiisopropylphospharamidite-9-D-ribofuranosyl-6-(2-(phenylthio)ethyl)purineon an Expedite (Millipore; Billerica, Mass.) RNA synthesizer using theconditions recommended by the manufacturers.

After the synthesis, the oligonucleotides are cleaved from the solidsupport by treatment with ammonium hydroxide (30%, 12 hours) and driedusing a vacuum centrifuge. The crude 2′-TOM protected RNA (1 μmol) istreated with a solution of magnesium monoperphthalate (MMPP; 2.9 μmoles)in carbonate buffer (10 mM, pH 10; 50 μL), followed by treatment with10N sodium hydroxide (0.15 mL). The solution is neutralized with aceticacid and then evaporated to dryness. The 2′-TOM-protected 2-amino-6vinyl purine containing oligonucleotide is dissolved in DMSO (100 μL)and treated with triethylamine (65 μL), followed by triethylammoniumtrihydrofluoride (75 μL), and incubated at 65° C. for 2 hours. FIG. 7illustrates these steps. The deprotected RNA is precipitated withbutanol, washed once with 100% cold ethanol and once with 70%ethanol/water.

The pellet is dissolved in 0.1 mL of diethylpyrocarbonate(DEPC)-treatedwater and purified by ion exchange HPLC on a Dionex (Sunnyvale, Calif.)DNA-pac column using a gradient of 0.01 M NaCl to 1 M NaCl containingEDTA in nuclease-free water. The major fraction is collected anddesalted in an OPC purification cartridge (ABI; Carlsbad, Calif.) usingthe conditions recommended by the manufacturer.

The 2-amino-6 vinyl-modified oligonucleotide is ligated onto the end ofan mRNA preparation containing the sequences of interest by splintligation, in which the mRNA and activated oligonucleotide are annealedto a DNA oligonucleotide containing sequences complementary to both themRNA and the activated oligonucleotide and are joined together using T4DNA ligase (New England BioLabs, Ipswich, Mass.). The resulting mRNA isthen subjected to in vitro translation and tRNA crosslinking.

Example 7 Synthesis of Linking tRNA Molecules Containing an Activated4-thiouridine Residue

A linking tRNA comprising an activated ^(4S)U moiety can be synthesizedfrom an oligonucleotide prepared as described in Example 1. A first RNAoligonucleotide comprising the 5′ portion of the tRNA (extending fromthe 5′ end to the last base of the anticodon) is synthesized, wherein a^(4S)U residue is incorporated at the desired position in the anticodon.The first oligonucleotide is annealed to a second RNA oligonucleotide,corresponding to the 3′ portion of the tRNA, that begins with the firstbase 3′ of the anticodon and extends to the base preceding the terminalA of the mature tRNA. The second RNA may be produced by chemicalsynthesis or by in vitro transcription of an appropriate DNA sequence.The first and second oligonucleotides are ligated together using T4 DNAligase (New England BioLabs, Ipswich, Mass.). To facilitate ligation, a20-nucleotide bridging or splint DNA that is complementary to 10 baseson each side of the junction is annealed to the two RNA oligonucleotidesprior to ligation. The ligation product is denatured by heating andpurified by polyacrylamide gel electrophoresis in the presence of 8 Murea. The synthesis of tRNA molecules from subfragments has beendescribed (e.g., U.S. Pat. Nos. 6,962,781; 7,351,812; 7,410,761; and7,488,600). 3′-Amino-3′-deoxyadensoine is prepared using known methodsand attached to the ligation product purified above using tRNAnucleotidyl transferase (Fraser, T. H. and Rich, A., Proc. Natl. Acad.Sci. U.S.A. 70:2671-2675, 1973; Merryman, C. et al., (2002). Chem, Biol.9:741-746, and references therein). Alternatively, a dinucleotideconsisting of cytidine-3′-amino-3′-deoxyadenosine or a trinucleotideconsisting of 5′-phospho-cytidine-cytidine-3′-amino-3′-deoxyadenosinecan be chemically synthesized (Zhang, B., Zhang, L., Sun, L., Cui, Z.(2002) Org. Lett. 4:3615-3618, 2002) and used as a replacement of thecorresponding terminal nucleotides of the mature tRNA via ligation withT4 RNA ligase. The resulting 3′-amino-terminated tRNA can be chargedwith an amino acid using the appropriate aminoacyl tRNA synthetase(Fraser, T. H. et al., op. cit.; Merryman, C. et al., op. cit.).Finally, the amidated tRNA is activated withN,N′-bis-bromoacetyl-1,2-diaminobenzene as described in Example 1.Alternatively, it is activated prior to or after ligation of the 5′ and3′ tRNA fragments.

The 3′ tRNA fragment can also be synthesized with puromycin replacingthe 3′ terminal adenosine using standard phosphoramidite methods(available from IDT, Coralville, Iowa). The resulting tRNA contains anamide-linked O-methyltyrosine at its 3′ end.

Example 8 Synthesis of3′-amino-5′-DMT-2′-TOM-N-6-Benzoyl-3′-deoxyadenosine

3′-Azido-3′-deoxyadenosine is synthesized as described by Robins et al.(J. Org. Chem. 66:8204-8210, 2001). 3′-azido-3′-deoxyguanosine (2.92 g;10 mmol) is dissolved in anhydrous pyridine (60 mL) with vigorousstirring under an argon atmosphere and cooled on an ice bath, followedby treatment with trimethylsylil chloride (3 molar equivalents; mol eq),and stirring for 60 minutes at room temperature. The resulting solutioncontaining the sylilated nucleoside is cooled on an ice bath and treatedwith benzoyl chloride (1.2 mol eq), allowed to reach room temperature,and then stirred for 4 hours. Water (10 mL) is added and the solution isstirred for 10 minutes. Concentrated ammonium hydroxide (20 mL) is addedand the reaction mixture is stirred for 15 minutes at room temperature.The resulting mixture was then evaporated to dryness under reducedpressure. The dried residue is stirred with cold water, filtered, anddried under high vacuum with phosphorus pentoxide, yielding3′-azido-6-N-benzoyl 3′-deoxyadenosine.

A dimethoxytrityl (DMT) protecting group is added at the 5′ position ofthe ribose moiety to produce3′-azido-5′-DMT-6-N-benzoyl-3′-deoxyadenosine. 3′-Azido-6-N-benzoyl3′-deoxyadenosine (8 mmol) is dissolved in anhydrous pyridine (80 mL)and treated with DMT-Cl (1.1 mol eq) and stirred at room temperature for6 hours. Water (5 mL) is added and the solution stirred for additional10 minutes. Volatiles are evaporated under reduced pressure and theresidue dissolved in dichloromethane (150 mL) and washed twice with asaturated solution of sodium bicarbonate (2×50 mL), water (50 mL), andbrine (50 mL). The organic residue is dried with magnesium sulfate,filtered, and evaporated under reduced pressure. The obtained solid iscrushed with ethyl ether and filtered to obtain3′-azido-5′-DMT-6-N-benzoyl-3′-deoxyadenosine, which is used in the nextstep without purification.

3′-Azido-5′-DMT-6-N-benzoyl-3′-deoxyadenosine is further modified byaddition of a 2′ (triisopropylsilyl)oxy]methyl (TOM) group to produce3′-azido-2′-TOM-5′-DMT-6-N-benzoyl-3′-deoxyadenosine.

3′-Azido-5′-DMT-6-N-benzoyl-3′-deoxyadenosine (7.8 mmol) is suspended indichloroethane (100 mL) containing diisopropylethylamine (1.5 mol eq)and cooled on an ice bath. [(Triisopropylsilyl)oxy]methyl chloride (1.1mol eq) is added to the solution and stirred for 60 minutes at roomtemperature, at which point water (2 mL) is added and stirred for 5minutes. Volatiles are evaporated under reduced pressure and the residuedissolved in dichloromethane (150 mL), washed twice with a saturatedsolution of sodium bicarbonate (2×50 mL), and washed once with brine (50mL). The organics are dried with magnesium sulfate, filtered, andevaporated under reduced pressure. The resulting solid is purified bycolumn chromatography on silica gel using dichloromethane/methanol(97:3) as the mobile phase. Fractions containing the desired product areevaporated under reduced pressure.

The azido functionality in3′-azido-2′-TOM-5′-DMT-6-N-benzoyl-3′-deoxyadenosine is reduced to anamine residue via catalytic hydrogenation with palladium3′-azido-2′-TOM-5′-DMT-6-N-benzoyl-3′-deoxyadenosine (6 mmol) isdissolved in ethanol (120 mL) containing 10% Pd/C (0.6 mol eq) andtreated with hydrogen gas (1 atm) for 12 hours. The solution is filteredthrough a pad of celite and the celite washed with ethanol (60 mL). Thesolvent is evaporated and the resulting white powder dried under highvacuum to yield 3′-amino-5′-DMT-2′-TBDMS-N-6-benzoyl-3′-deoxyadenosine.

Example 9 Solid Support Immobilization of3′-amino-5′-DMT-2′-TBDMS-N-6-benzoyl-3′-deoxyadenosine

The amino-containing nucleoside is covalently linked to a controlledpored glass solid phase synthesis support using the general procedurereported by Eisenhut and Richert, J. Org. Chem. 74:26-37, 2009.

Example 10 Solid Phase Synthesis of Amino-terminated TrinucleotidepCpCpA-3′-NH₂

Trinucleotide pCpCpA-3′-NH₂ is synthesized on a 5-micromole scale viasolid phase synthesis on CPG-immobilized3′-amino-5′-DMT-2′-TBDMS-N-6-benzoyl-3′-deoxyadenosine using standardphosphoramidite chemistry. The synthesis is performed on an Expedite(Millipore; Billerica, Mass.) RNA synthesizer using the conditionsrecommended by the manufacturers. After the synthesis, the trinucleotideis cleaved from the solid support by treatment with ammonium hydroxide(30%, 12 hours) and dried using a vacuum centrifuge. The2′-TOM-protected trinucleotide is dissolved in DMSO (100 μL) and treatedwith triethylamine (65 μL), followed by tributylammoniumtrihydrofluoride (75 μL), and incubated at 65° C. for 6 hours. Thesample is diluted in water (20 mL), lyophilized, and purified bypreparative HPLC on a C₁₈ reverse phase column using a linear gradientof 0-30% methanol in triethylammonium bicarbonate (10 mM). Fractionscontaining the desired product are lyophilized to dryness.

The 3′-amino modified trinucleotide is ligated onto the end of an mRNApreparation containing the sequences of interest by splint ligation, inwhich the mRNA and activated oligo are annealed to a DNA oligonucleotidecontaining sequences complementary to both the mRNA and the activatedoligonucleotide, and joined together using T4 DNA ligase (New EnglandBioLabs, Ipswich, Mass.).

Example 11 Preparation of Azido-modified Oligonucleotides from 3′-amino3′-deoxyadenosine-containing Oligonucleotides

An oligonucleotide (5 micromoles) containing a single terminal 3′-aminonucleoside as described in Example 10 is dissolved in 50 μL of 70 mMboric acid (pH 8.5) and cooled on an ice-bath. Formamide (30 μL) isadded, followed by addition of a freshly prepared solution ofN-(5-azido-2-nitrobenzoyloxy)succinimide (15 molar equivalents)(Sigma-Aldrich, St. Louis, Mo.) in DMF (40 μL) (Handbook of RNABiochemistry; Hartmann, R. K., Bindereif, A., Schon, A., and Westhof, E.M., Eds., 2005, Wiley-VCH). The resulting solution is incubated for 60minutes on ice and a second aliquot of the NHS-azido ester is added andincubated for an additional hour. The solution is extracted twice withchloroform (300 μL) at room temperature and precipitated by addition of20 μL of 3 M sodium acetate and 1.3 mL ethanol. The mixture is cooled to−70° C. for 1 hour and the precipitated oligonucleotide pelleted bycentrifugation. The oligonucleotide is washed once with 70% ethanol,centrifuged, and evaporated to dryness using a Speed-Vac lyophilizer.The oligonucleotide is purified by preparative reverse phase HPLC on aC-18 column using a linear gradient of triethylammonium acetate (20 mM)to 60% acetonitrile in triethylammonium acetate (20 mM). Fractionscontaining the oligonucleotide are lyophilized to dryness. The resultingmRNA is then subjected to in vitro translation reaction and crosslinkedto its peptide target by either one of the two procedures describedbelow.

Example 12 Photocrosslinking of Azido-containing mRNA and an Amino AcidSide Chain at the Peptidyl Transfer Center

An mRNA containing a single terminal azido modification is subjected toan in vitro translation reaction and crosslinked to its peptide targetby irradiation with UV light using a Rayonet RPR100 photoreactor at 4°C. for 2 hours. Crosslinked peptide-mRNA fusions are isolated bytreating the translation mixture with a release buffer (1M NaCl, 20 mMEDTA, 0.1 Bicine, pH 8.3) and then by oligonucleotide affinitypurification on an oligo-dT containing column.

Example 13 Crosslinking of Azido-containing mRNA and anAlkyne-containing Amino Acid Side Chain at the Peptidyl Transfer CenterVia Copper-catalyzed Huisgen Cycloaddition

An mRNA containing a single terminal azido modification is subjected toan in vitro translation reaction and crosslinked to its peptide target,which contains an amino acid with an alkynyl side chain at the peptidyltransfer center, by addition of a Cu(I) containing salt and incubationat 4° C. for 10 hours (Neumann, H. et al., Nature, 464, 441-444, 2010).Crosslinked peptide-mRNA fusions are isolated by treating thetranslation mixture with a release buffer (1M NaCl, 20 mM EDTA, 0.1Bicine, pH 8.3) and then by oligonucleotide affinity purification on anoligo-dT containing column.

What is claimed is:
 1. A method for linking an mRNA molecule to apolypeptide, the method comprising: (a) providing an mRNA moleculecomprising a reactive nucleoside that is reactive with a crosslinker,wherein the crosslinker is an S-4 alkylated thiouridine comprisingN-(2-acetamidophenyl)-2-bromoacetamide at the S-4 position of thealkylated thiouridine; (b) providing a translation system comprising alinking aminoacyl-tRNA comprising an amino acid residue linked to alinking tRNA by an amide bond, wherein the linking tRNA comprises ananticodon comprising the crosslinker; (c) translating the mRNA moleculein the translation system to produce a polypeptide into which the aminoacid residue, still linked to the tRNA, is incorporated; and (d) duringor after step (c), crosslinking the reactive nucleoside of the mRNAmolecule to the crosslinker of the anticodon of the linking tRNA,thereby linking the mRNA molecule to the polypeptide through the linkingtRNA.
 2. The method of claim 1, wherein the linking tRNA is a suppressortRNA.
 3. The method of claim 1, wherein the translation system is apurified in vitro translation system.
 4. A method for linking an mRNAmolecule to a polypeptide, the method comprising: (a) providing an mRNAmolecule comprising a reactive nucleoside that is reactive with acrosslinker, wherein the crosslinker is an S-4-alkylated thiouridinecomprising at the S-4 position: (i)N-(3-acetamidophenyl)-2-bromoacetamide;(ii)N-(4-acetamidophenyl)-2-bromoacetamide; (iii)N-((2-acetamidomethyl)benzyl)-2-bromoacetamide; (iv)N-((3-acetamidomethyl)benzyl)-2-bromoacetamide; (v)N-((4-acetamidomethyl)benzyl)-2-bromoacetamide; (vi)(Z/E)-N-(4-acetamidobut-2-enyl)-2-bromoacetamide; or (vii)(Z/E)-N-(2-acetamidovinyl)-2-bromoacetamide; (b) providing a translationsystem comprising a linking aminoacyl-tRNA comprising an amino acidresidue linked to a linking tRNA by an amide bond, wherein the linkingtRNA comprises an anticodon comprising the crosslinker; (c) translatingthe mRNA molecule in the translation system to produce a polypeptideinto which the amino acid residue, still linked to the tRNA, isincorporated; and (d) during or after step (c), crosslinking thereactive nucleoside of the mRNA molecule to the crosslinker of theanticodon of the linking tRNA, thereby linking the mRNA molecule to thepolypeptide through the linking tRNA.
 5. The method of claim 4, whereinthe linking tRNA is a suppressor tRNA.
 6. The method of claim 4, whereinthe translation system is a purified in vitro translation system.